scholarly journals Expression of the recombinant C-terminal of the S1 domain and N-terminal of the S2 domain of the spike protein of porcine epidemic diarrhea virus

2021 ◽  
pp. 2913-2918
Author(s):  
Jiraporn Sritun ◽  
Natnaree Inthong ◽  
Siriluk Jala ◽  
Sakuna Phatthanakunanan ◽  
Khomson Satchasataporn ◽  
...  
Keyword(s):  
Induction Time ◽  
Porcine Epidemic Diarrhea Virus ◽  
Polyclonal Antibodies ◽  
Optimum Concentration ◽  
Economic Losses ◽  
S Protein ◽  
Thai Isolate ◽  
Diarrhea Virus ◽  
S Gene ◽  
Porcine Epidemic Diarrhea

Background and Aim: Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in suckling piglets, leading to severe economic losses in the swine industry. Commercial vaccines have limited effectiveness against different genogroups of PEDV and the shedding of virus. The C-terminal of the S1 domain and the N-terminal of the S2 domain (S1-2) protein of the spike (S) protein have four neutralizing epitopes. However, research on the expression of the S1-2 segment of the S gene has been limited. In this study, we expressed a recombinant S1-2 protein of the S protein of the PEDV Thai isolate and characterized the immunological properties of the recombinant S1-2 protein. Materials and Methods: The S1-2 segment of the S gene of the PEDV Thai isolate (G2b) was amplified, cloned into the pBAD202/D-TOPO® vector (Invitrogen, Carlsbad, CA, USA), and expressed in Escherichia coli. The optimum concentration of arabinose and the optimum induction time for the expression of the recombinant S1-2 protein were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenic reactivity of the recombinant S1-2 protein was determined using Western blot analysis with rabbit polyclonal antibodies against the SM98 strain of PEDV (G1a). Results: The recombinant S1-2 segment of the S gene of the PEDV Thai isolate protein was cloned and the recombinant S1-2 protein was successfully expressed. The optimum concentration of arabinose and the optimum induction time for the induction of the recombinant S1-2 protein were 0.2% and 8 h, respectively. The recombinant S1-2 protein reacted specifically with both rabbit anti-histidine polyclonal antibodies and rabbit anti-PEDV polyclonal antibodies. Conclusion: The recombinant S1-2 protein reacted with rabbit anti-PEDV polyclonal antibodies induced by the different PEDV genogroup. Therefore, the recombinant S1-2 protein may be a useful tool for the development of a diagnostic test for PEDV or for a vaccine against PEDV.

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 Successive Passage In Vitro Led to Lower Virulence and Higher Titer of A Variant Porcine Epidemic Diarrhea Virus

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 391 ◽  
Author(s):  
Pengwei Zhao ◽  
Song Wang ◽  
Zhi Chen ◽  
Jiang Yu ◽  
Rongzhi Tang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Porcine Epidemic Diarrhea Virus ◽  
Vero Cells ◽  
Economic Losses ◽  
Diarrhea Virus ◽  
S Gene ◽  
Successive Passage ◽  
Porcine Epidemic Diarrhea

A highly virulent porcine epidemic diarrhea virus (PEDV) appeared in China and spread rapidly to neighbor countries, which have led to great economic losses to the pig industry. In the present study, we isolated a PEDV using Vero cells and serially propagated 100 passages. PEDV SDSX16 was characterized in vitro and in vivo. The viral titers increased to 107.6 TCID50/mL (100th) by serial passages. The spike (S) gene and the whole gene of the SDSX16 virus was fully sequenced to assess the genetic stability and relatedness to previously identified PEDV. Along with successive passage in vitro, there were 18 nucleotides (nt) deletion occurred in the spike (S) gene resulting in a deletion of six amino acids when the SDSX16 strain was passaged to the 64th generation, and this deletion was stable until the P100. However, the ORF1a/b, M, N, E, and ORF3 genes had only a few point mutations in amino acids and no deletions. According to growth kinetics experiments, the SDSX16 deletion strain significantly enhanced its replication in Vero cells since it was passaged to the 64th generation. The animal studies showed that PEDV SDSX16-P10 caused more severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis than SDSX16-P75, indicating that SDSX16-P10 is enteropathogenic in the natural host, and the pathogenicity of SDSX16 decreased with successive passage in vitro. However, SDSX16-P10 was found to cause lower levels of cytokine expression than SDSX16-P75 using real-time PCR and flow cytometry, such as IL1β, IL6, IFN-β, TNF-α, indicating that SDSX16-P10 might inhibit the expression of cytokines. Our data indicated that successive passage in vitro resulted in virulent attenuation in vivo of the PEDV variant strain SDSX16.

scholarly journals The S Gene Is Necessary but Not Sufficient for the Virulence of Porcine Epidemic Diarrhea Virus Novel Variant Strain BJ2011C

2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Di Wang ◽  
Xinna Ge ◽  
Dongjie Chen ◽  
Jie Li ◽  
Yueqi Cai ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Vaccine Development ◽  
Economic Losses ◽  
Avirulent Strain ◽  
Diarrhea Virus ◽  
Severe Diarrhea ◽  
S Gene ◽  
Viral Virulence ◽  
Porcine Epidemic Diarrhea ◽  
Highly Virulent

ABSTRACT The recently emerged highly virulent variants of porcine epidemic diarrhea virus (PEDV) have caused colossal economic losses to the worldwide swine industry. In this study, we investigated the viral virulence determinants by constructing a series of chimeric mutants between the highly virulent strain BJ2011C and the avirulent strain CHM2013. When tested in the 2-day-old piglet model, wild-type (WT) BJ2011C caused severe diarrhea and death of the piglets within 72 h. In contrast, its chimeric derivative carrying the S gene from CHM2013 (BJ2011C-S CHM ) was avirulent to the piglets. Moreover, reciprocal substitution of the BJ2011C S gene (CHM2013-S BJ ) did not enable CHM2013 to gain any virulence. However, when the whole structural protein-coding region of BJ2011C (CHM2013-SP BJ ) was swapped, CHM2013 started to gain the ability to efficiently colonize the intestinal tract and caused diarrhea in piglets. A further gain of virulence required additional acquisition of the 3′ untranslated region (UTR) of BJ2011C, and the resultant virus (CHM2013-SP + 3UTR BJ ) caused more severe diarrhea and death of piglets. Together, our findings suggest that the virulence of PEDV epidemic strains is a multigenic event and that the S gene is only one of the necessary determinants. IMPORTANCE The recently emerged highly virulent PEDV variants are the major cause of the global porcine epidemic diarrhea (PED) pandemic. The S gene of the variants undergoes remarkable variations and has been thought to be the virulence determinant for the enhanced pathogenesis. Our studies here showed that the S gene is only part of the story and that full virulence requires cooperation from other genes. Our findings provide insight into the pathogenic mechanism of the highly virulent PEDV variants and have implications for future vaccine development.

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 Deletion of both the Tyrosine-Based Endocytosis Signal and the Endoplasmic Reticulum Retrieval Signal in the Cytoplasmic Tail of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Pigs

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Yixuan Hou ◽  
Tea Meulia ◽  
Xiang Gao ◽  
Linda J. Saif ◽  
Qiuhong Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Vero Cell ◽  
Porcine Epidemic Diarrhea Virus ◽  
Cytoplasmic Tail ◽  
S Protein ◽  
Diarrhea Virus ◽  
Neonatal Piglets ◽  
Porcine Epidemic Diarrhea ◽  
Intracellular Sorting ◽  
S Proteins

ABSTRACTPorcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets. The PEDV spike (S) protein contains two intracellular sorting motifs, YxxΦ (tyrosine-based motif YEVF or YEAF) and KVHVQ at the cytoplasmic tail, yet their functions have not been fully elucidated. Some Vero cell-adapted and/or attenuated PEDV variants contain ablations in these two motifs. We hypothesized that these motifs contribute to viral pathogenicity. By transiently expressing PEDV S proteins with mutations in the motifs, we confirmed that the motif KVHVQ is involved in retention of the S proteins in the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). In addition, we showed that the YxxΦ motif triggers endocytosis of S proteins. These two motifs synergistically regulate the level of S expressed on the cell surface. To investigate their role in viral pathogenicity, we generated three recombinant PEDVs by introducing deletions or a mutation in the two motifs of the infectious clone of PEDV PC22A strain (icPC22A): (i) icΔ10aa (ΔYxxΦEKVHVQ), (ii) icΔ5aa (ΔKVHVQ), and (iii) icYA (Y1378A, to an inactivated motif, AEVF). Infection of Vero cells with icΔ10aa resulted in larger syncytia and more virions, with reduced numbers of S protein projections on the surface compared with icPC22A. Furthermore, we orally inoculated five groups of 5-day-old gnotobiotic piglets with the three mutants, icPC22A, or a mock treatment. Mutant icΔ10aa caused less severe diarrhea rate and significantly milder intestinal lesions than icPC22A, icΔ5aa, and icYA. These data suggest that the deletion of both motifs can reduce the virulence of PEDV in piglets.IMPORTANCEMany coronaviruses (CoVs) possess conserved motifs YxxΦ and/or KxHxx/KKxx in the cytoplasmic tail of the S protein. The KxHxx/KKxx motif has been identified as the ER retrieval signal, but the function of the YxxΦ motif in the intracellular sorting of CoV S proteins remains controversial. In this study, we showed that the YxxΦ of PEDV S protein is an endocytosis signal. Furthermore, using reverse genetics technology, we evaluated its role in PEDV pathogenicity in neonatal piglets. Our results explain one attenuation mechanism of Vero cell-adapted PEDV variants lacking functional YxxΦ and KVHVQ motifs. Knowledge from this study may aid in the design of efficacious live attenuated vaccines against PEDV, as well as other CoVs bearing the same motif in their S protein.

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 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.

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