scholarly journals S1 Subunit of Spike Protein from a Current Highly Virulent Porcine Epidemic Diarrhea Virus is an Important Determinant of Virulence in Piglets

2018 ◽  
Author(s):  
Tohru Suzuki ◽  
Yutaka Terada ◽  
Luis Enjuanes ◽  
Seiichi Ohashi ◽  
Wataru Kamitani
Keyword(s):  
North American ◽  
Porcine Epidemic Diarrhea Virus ◽  
Reverse Genetics ◽  
Important Determinant ◽  
Animal Health ◽  
Watery Diarrhea ◽  
S Protein ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea ◽  
Highly Virulent

Base on the sequence of S genes, which encode spike proteins, we previously identified three different types (North American, S INDEL and S large-DEL types) of porcine epidemic diarrhea virus (PEDV) that have re-emerged in Japan since 2013. Base on experimental infections with the North American and S large-DEL types, we also hypothesized that PEDV virulence may be linked to the S1 subunit of the S protein. To test this hypothesis, we have now assayed in gnotobiotic piglets various recombinant PEDVs generated by reverse genetics. Piglets inoculated with CV777 maintained in National Institute of Animal Health, along with piglets infected with a recombinant form of the same virus, developed subclinical to mild diarrhea. In contrast, severe watery diarrhea, dehydration, weight loss, astasia, and high mortality were observed in piglets inoculated with recombinant strains in which the S gene was partially or fully replaced with corresponding sequences from the highly virulent Japanese PEDV isolate OKN-1/JPN/2013. Indeed, symptoms resembled those in piglets inoculated with the OKN-1/JPN/2013, and were especially pronounced in younger piglets. Collectively, the data demonstrate that the S1 subunit of the S protein is an important determinant of PEDV virulence, and advance development of new vaccine candidate.

scholarly journals S1 Subunit of Spike Protein from a Current Highly Virulent Porcine Epidemic Diarrhea Virus Is an Important Determinant of Virulence in Piglets

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 467 ◽  
Author(s):  
Tohru Suzuki ◽  
Yutaka Terada ◽  
Luis Enjuanes ◽  
Seiichi Ohashi ◽  
Wataru Kamitani
Keyword(s):  
North American ◽  
Porcine Epidemic Diarrhea Virus ◽  
Reverse Genetics ◽  
Important Determinant ◽  
Animal Health ◽  
Watery Diarrhea ◽  
S Protein ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea ◽  
Highly Virulent

Base on the sequence of S genes, which encode spike proteins, we previously identified three different types (North American, S INDEL, and S large-DEL types) of porcine epidemic diarrhea virus (PEDV) that have re-emerged in Japan since 2013. Based on experimental infections with the North American and S large-DEL types, we also hypothesized that PEDV virulence may be linked to the S1 subunit of the S protein. To test this hypothesis, we have now assayed in gnotobiotic piglets various recombinant PEDVs generated by reverse genetics. Piglets inoculated with CV777 maintained in National Institute of Animal Health, along with piglets infected with a recombinant form of the same virus, developed subclinical to mild diarrhea. In contrast, severe watery diarrhea, dehydration, weight loss, astasia, and high mortality were observed in piglets inoculated with recombinant strains in which the S gene was partially or fully replaced with corresponding sequences from the highly virulent Japanese PEDV isolate OKN-1/JPN/2013. Indeed, symptoms resembled those in piglets inoculated with the OKN-1/JPN/2013, and were especially pronounced in younger piglets. Collectively, the data demonstrate that the S1 subunit of the S protein is an important determinant of PEDV virulence, and advance development of new vaccine candidate.

scholarly journals Deletion of a 197-Amino-Acid Region in the N-Terminal Domain of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Piglets

2017 ◽  
Vol 91 (14) ◽  
Author(s):  
Yixuan Hou ◽  
Chun-Ming Lin ◽  
Masaru Yokoyama ◽  
Boyd L. Yount ◽  
Douglas Marthaler ◽  
...  
Keyword(s):  
Amino Acid ◽  
Porcine Epidemic Diarrhea Virus ◽  
Protective Immunity ◽  
Cross Protection ◽  
Spike Protein ◽  
Virulence Determinants ◽  
S Protein ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea ◽  
Highly Virulent

ABSTRACT We previously isolated a porcine epidemic diarrhea virus (PEDV) strain, PC177, by blind serial passaging of the intestinal contents of a diarrheic piglet in Vero cell culture. Compared with the highly virulent U.S. PEDV strain PC21A, the tissue culture-adapted PC177 (TC-PC177) contains a 197-amino-acid (aa) deletion in the N-terminal domain of the spike (S) protein. We orally inoculated neonatal, conventional suckling piglets with TC-PC177 or PC21A to compare their pathogenicities. Within 7 days postinoculation, TC-PC177 caused mild diarrhea and lower fecal viral RNA shedding, with no mortality, whereas PC21A caused severe clinical signs and 55% mortality. To investigate whether infection with TC-PC177 can induce cross-protection against challenge with a highly virulent PEDV strain, all the surviving piglets were challenged with PC21A at 3 weeks postinoculation. Compared with 100% protection in piglets initially inoculated with PC21A, 88% and 100% TC-PC177- and mock-inoculated piglets had diarrhea following challenge, respectively, indicating incomplete cross-protection. To investigate whether this 197-aa deletion was the determinant for the attenuation of TC-PC177, we generated a mutant (icPC22A-S1Δ197) bearing the 197-aa deletion from an infectious cDNA clone of the highly virulent PEDV PC22A strain (infectious clone PC22A, icPC22A). In neonatal gnotobiotic pigs, the icPC22A-S1Δ197 virus caused mild to moderate diarrhea, lower titers of viral shedding, and no mortality, whereas the icPC22A virus caused severe diarrhea and 100% mortality. Our data indicate that deletion of this 197-aa fragment in the spike protein can attenuate a highly virulent PEDV, but the virus may lose important epitopes for inducing robust protective immunity. IMPORTANCE The emerging, highly virulent PEDV strains have caused substantial economic losses worldwide. However, the virulence determinants are not established. In this study, we found that a 197-aa deletion in the N-terminal region of the S protein did not alter virus (TC-PC177) tissue tropism but reduced the virulence of the highly virulent PEDV strain PC22A in neonatal piglets. We also demonstrated that the primary infection with TC-PC177 failed to induce complete cross-protection against challenge by the highly virulent PEDV PC21A, suggesting that the 197-aa region may contain important epitopes for inducing protective immunity. Our results provide an insight into the role of this large deletion in virus propagation and pathogenicity. In addition, the reverse genetics platform of the PC22A strain was further optimized for the rescue of recombinant PEDV viruses in vitro. This breakthrough allows us to investigate other virulence determinants of PEDV strains and will provide knowledge leading to better control PEDV infections.

scholarly journals Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Akitsu Masuda ◽  
Jae Man Lee ◽  
Takeshi Miyata ◽  
Takeru Ebihara ◽  
Kohei Kakino ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Vector System ◽  
Watery Diarrhea ◽  
Small Scale ◽  
Efficient Production ◽  
S Protein ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

AbstractPorcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.

scholarly journals The Accessory Protein ORF3 Contributes to Porcine Epidemic Diarrhea Virus Replication by Direct Binding to the Spike Protein

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 399 ◽  
Author(s):  
Challika Kaewborisuth ◽  
Qigai He ◽  
Anan Jongkaewwattana
Keyword(s):  
Virus Replication ◽  
Porcine Epidemic Diarrhea Virus ◽  
Watery Diarrhea ◽  
Accessory Protein ◽  
Structural Protein ◽  
S Protein ◽  
Diarrhea Virus ◽  
Neonatal Piglets ◽  
Infected Cells ◽  
Porcine Epidemic Diarrhea

The porcine epidemic diarrhea virus (PEDV) is an important swine pathogen responsible for severe watery diarrhea, particularly in neonatal piglets. Despite extensive studies performed to elucidate the function of several viral proteins, the contribution of an accessory protein ORF3 in PEDV replication is still largely unknown. Here, we constructed expression plasmids as well as recombinant PEDV carrying myc-tagged ORF3 to assess their expression and subcellular localization in both transfected and infected cells. In PEDV-infected cells, ORF3 was predominantly localized in the cytoplasm, partially in the endoplasmic reticulum (ER) and the Golgi apparatus (Golgi). Interestingly, ORF3 with the N-terminal Flag tag was also detected on the cell surface concomitant with the spike (S) protein as determined by flow cytometry and confocal microscopy. ORF3 and S proteins were also co-localized at perinuclear compartments and in the vesicle-like structures in transfected and infected cells. We also demonstrated that both full-length and naturally truncated ORF3 proteins could interact with the S protein but with different binding affinity, which correlate with the ability of the protein to regulate virus replication in cell culture. Collectively, our results underscore the unprecedented role of the ORF3, which involves the interaction of ORF3 with S and, possibly, other structural protein during PEDV replication.

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 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 Deciphering the biology of porcine epidemic diarrhea virus in the era of reverse genetics

2016 ◽  
Vol 226 ◽  
pp. 152-171 ◽  
Author(s):  
Samaporn Teeravechyan ◽  
Phanramphoei Namprachan Frantz ◽  
Phonphimon Wongthida ◽  
Thanathom Chailangkarn ◽  
Peera Jaru-ampornpan ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Reverse Genetics ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

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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