scholarly journals The N-terminal domain of Spike protein contributes to antigenicity difference between porcine epidemic diarrhea virus genotypes G1 and G2

2021 ◽  
Author(s):  
Qi-long Qiao ◽  
Ning Li ◽  
Ming-zhen Song ◽  
Jing Chen ◽  
Pan pan Yang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Porcine Epidemic Diarrhea Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
S Protein ◽  
Diarrhea Virus ◽  
Terminal Domain ◽  
Porcine Epidemic Diarrhea ◽  
Amino Acid Mutations ◽  
Multiple Amino Acid ◽  
S Proteins

Porcine epidemic diarrhea virus (PEDV) strains have been clarified into two genotypes, G1 and G2, based on the sequence of the spike (S) gene. Amino acid mutations that distinguish the two PEDV genotypes were mostly located in the N-terminal domain (NTD) (aa 1-380) of S protein. The fact of increased outbreaks of G2 subtype PEDV and the failure of G1 subtype PEDV strain (CV777)-based vaccine in China since 2010 suggested that multiple amino acid mutations located in the NTD altered the antigenicity of S protein. To determine the role of the NTD of S protein in the antigenicity difference, the NTD of the CV777 vaccine strain (G1) and CH/ZMDZY/11 strain (G2) was expressed in E. coli, respectively. polyclonal antibodies (PAbs) against genotype-specific S proteins were prepared by immunizing BALB/c mice using purified S proteins. Antigenicity was systematically compared by detection of PAbs against two genotype PEDV strains and purified S proteins using Western blot, indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and serum cross-neutralization assay (SN). Consistent with the multiple amino acid mutations in the NTD of S protein, different antigenic cross-reactivity between the two genotypes was demonstrated. There was six-fold and more than twenty-fold difference in ELISA and SN titer between anti-CV777 S protein antibodies against G1 and G2 subtype strains, respectively. There was twofold and eight-fold difference in ELISA and SN titer between anti-ZMDZY S protein antibodies against G1 and G2 genotype strains, respectively. The results proved that the NTD of S protein contributes to the antigenicity difference between PEDV genotypes G1 and G2, and highlighted a G2 strain should be used to develop a vaccine for providing better protection against prevalent genotype of PEDV.

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 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 Changes in the spike and nucleocapsid protein of porcine epidemic diarrhea virus strain in Vietnam—a molecular potential for the vaccine development?

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12329
Author(s):  
Thach Xuan Tran ◽  
Nguyen T.K. Lien ◽  
Ha T. Thu ◽  
Nguyen Dinh Duy ◽  
Bui T.T. Duong ◽  
...  
Keyword(s):  
Amino Acid ◽  
Porcine Epidemic Diarrhea Virus ◽  
Vaccine Development ◽  
Effective Vaccine ◽  
Host Immune System ◽  
S Protein ◽  
Orf3 Protein ◽  
N Protein ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Background Porcine epidemic diarrhea virus (PEDV) is a dangerous virus causing large piglet losses. PEDV spread rapidly between pig farms and caused the death of up to 90% of infected piglets. Current vaccines are only partially effective in providing immunity to suckling due to the rapid dissemination and ongoing evolution of PEDV. Methods In this study, the complete genome of a PEDV strain in Vietnam 2018 (IBT/VN/2018 strain) has been sequenced. The nucleotide sequence of each fragment was assembled to build a continuous complete sequence using the DNASTAR program. The complete nucleotide sequences and amino acid sequences of S, N, and ORF3 genes were aligned and analyzed to detect the mutations. Results The full-length genome was determined with 28,031 nucleotides in length which consisted of the 5′UTR, ORF1ab, S protein, ORF3, E protein, M protein, N protein, and 3′UTR region. The phylogenetic analysis showed that the IBT/VN/2018 strain was highly virulent belonged to the G2b subgroup along with the Northern American and Asian S-INDEL strains. Multiple sequence alignment of deduced amino acids revealed numerous mutations in the S, N, and ORF3 regions including one substitution 766P > L766 in the epitope SS6; two in the S0subdomain (135DN136 > 135SI136 and N144> D144); two in subdomain SHR1 at aa 1009L > M1009 and 1089S > L1089; one at aa 1279P > S1279 in subdomain SHR2 of the S protein; two at aa 364N > I364 and 378N > S378 in the N protein; four at aa 25L > S25, 70I > V70, 107C > F107, and 168D > N168 in the ORF3 protein. We identified two insertions (at aa 59NQGV62 and aa 145N) and one deletion (at aa 168DI169) in S protein. Remarkable, eight amino acid substitutions (294I > M294, 318A > S318, 335V > I335, 361A > T361, 497R > T497, 501SH502 > 501IY502, 506I > T506, 682V > I682, and 777P > L777) were found in SA subdomain. Besides, N- and O-glycosylation analysis of S, N, and ORF3 protein reveals three known sites (25G+, 123N+, and 62V+) and three novel sites (144D+, 1009M+, and 1279L+) in the IBT/VN/2018 strain compared with the vaccine strains. Taken together, the results showed that mutations in the S, N, and ORF3 genes can affect receptor specificity, viral pathogenicity, and the ability to evade the host immune system of the IBT/VN/2018 strain. Our results highlight the importance of molecular characterization of field strains of PEDV for the development of an effective vaccine to control PEDV infections in Vietnam.

Development of an indirect ELISA based on a truncated S protein of the porcine epidemic diarrhea virus

2015 ◽  
Vol 61 (11) ◽  
pp. 811-817 ◽  
Author(s):  
Yufeng Li ◽  
Fangyuan Zheng ◽  
Baochao Fan ◽  
Hassan Mushtaq Muhammad ◽  
Yao Zou ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Indirect Elisa ◽  
Cross Reactivity ◽  
Virus Neutralization ◽  
Enzyme Linked Immunosorbent Assay ◽  
S Protein ◽  
Diarrhea Virus ◽  
Coefficients Of Variation ◽  
Porcine Epidemic Diarrhea ◽  
The Relationship

Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by the porcine epidemic diarrhea virus (PEDV). To find a suitable ELISA method to assess the infection of PEDV and the effectiveness of vaccines, we developed and evaluated an indirect enzyme-linked immunosorbent assay (iELISA) based on a truncated recombinant spike (S) protein expressed in Escherichia coli. The parameters of the iELISA were optimized, and the cutoff value determined as 0.259 by analyzing optical density (OD) values of 80 PEDV negative sera confirmed by western blot. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were both less than 10%. Cross-reactivity assays demonstrated that iELISA was PEDV-specific. A virus neutralization test with sera of 7 different OD values showed a positive correlation between the OD values and virus neutralization. The results suggest this iELISA is specific, sensitive, and repeatable. Further studies should focus on the relationship between OD values of sera and its virus neutralization.

scholarly journals Immunohistochemical Detection of Porcine Epidemic Diarrhea Virus Compared to Other Methods

1998 ◽  
Vol 5 (3) ◽  
pp. 412-414 ◽  
Author(s):  
Franco Guscetti ◽  
Curzio Bernasconi ◽  
Kurt Tobler ◽  
Kristien Van Reeth ◽  
Andreas Pospischil ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunofluorescence ◽  
Good Reliability ◽  
Diarrhea Virus ◽  
Pcr Method ◽  
Porcine Epidemic Diarrhea ◽  
Cryostat Sections ◽  
Formalin Fixed

ABSTRACT An immunohistochemistry method using formalin-fixed tissues, a direct immunofluorescence method using cryostat sections, an enzyme-linked immunosorbent assay (ELISA), and a PCR method were compared for diagnosis in a litter of weaned pigs that had been experimentally inoculated with wild-type porcine epidemic diarrhea virus (PEDV) and killed between 6 and 60 h after onset of diarrhea. The immunohistochemistry method proved to be as reliable as direct immunofluorescence for diagnosis of PEDV in tissues collected postmortem. The good reliability of ELISA for investigating clinical samples was confirmed, whereas the PCR method used was ineffective.

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.

scholarly journals Porcine Epidemic Diarrhea Virus ORF3 Protein Is Transported through the Exocytic Pathway

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Fusheng Si ◽  
Bingqing Chen ◽  
Xiaoxia Hu ◽  
Ruisong Yu ◽  
Shijuan Dong ◽  
...  
Keyword(s):  
Ion Channel ◽  
Porcine Epidemic Diarrhea Virus ◽  
Intracellular Trafficking ◽  
Expression Vectors ◽  
Orf3 Protein ◽  
Diarrhea Virus ◽  
Terminal Domain ◽  
Infected Cells ◽  
Porcine Epidemic Diarrhea ◽  
Exocytic Pathway

ABSTRACT Accessory genes occurring between the S and E genes of coronaviruses have been studied quite intensively during the last decades. In porcine epidemic diarrhea virus (PEDV), the only gene at this location, ORF3, encodes a 224-residue membrane protein shown to exhibit ion channel activity and to enhance virus production. However, little is known about its intracellular trafficking or about its function during PEDV infection. In this study, two recombinant PEDVs were rescued by targeted RNA recombination, one carrying the full-length ORF3 gene and one from which the gene had been deleted entirely. These viruses as well as a PEDV encoding a naturally truncated ORF3 protein were employed to study the ORF3 protein’s subcellular trafficking. In addition, ORF3 expression vectors were constructed to study the protein’s independent transport. Our results show that the ORF3 protein uses the exocytic pathway to move to and accumulate in the Golgi area of the cell similarly in infected and transfected cells. Like the S protein, but unlike the other structural proteins M and N, the ORF3 protein was additionally observed at the surface of PEDV-infected cells. In addition, the C-terminally truncated ORF3 protein entered the exocytic pathway but it was unable to leave the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartment (ERGIC). Consistently, a YxxØ motif essential for ER exit was identified in the C-terminal domain. Finally, despite the use of sensitive antibodies and assays no ORF3 protein could be detected in highly purified PEDV particles, indicating that the protein is not a structural virion component. IMPORTANCE Coronaviruses typically express several accessory proteins. They vary in number and nature, and only one is conserved among most of the coronaviruses, pointing at an important biological function for this protein. PEDV is peculiar in that it expresses just this one accessory protein, termed the ORF3 protein. While its analogs in other coronaviruses have been studied to different extents, and these studies have indicated that they share an ion channel property, little is still known about the features and functions of the PEDV ORF3 protein except for its association with virulence. In this investigation, we studied the intracellular trafficking of the ORF3 protein both in infected cells and when expressed independently. In addition, we analyzed the effects of mutations in five sorting motifs in its C-terminal domain and investigated whether the protein, found to follow the same exocytic route by which the viral structural membrane proteins travel, is also incorporated into virions.

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.

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