Evaluation of the Efficacy of a Recombinant Adenovirus Expressing the Spike Protein of Porcine Epidemic Diarrhea Virus in Pigs

In recent years, many studies have shown that recombinant adenovirus live vector-based vaccines are a promising novel vaccine candidate against virus infection. Therefore, in this study, a new type of recombinant adenovirus expressing the spike (S) protein of porcine epidemic diarrhea virus (PEDV), rAd-PEDV-S, was generated, and its characteristics were determined. Then, its efficacy as a vaccine candidate was evaluated in 4-week-old pigs. The results showed that the S protein could be well expressed at a high level in rAd-PEDV-S-infected cells and that the viral titers could reach 1011 PFU/mL. Further animal experimental results showed that rAd-PEDV-S elicited a significant PEDV-specific humoral immune response after vaccination (P<0.05). In addition, rAd-PEDV-S provided partial protection for pigs against the highly virulent PEDV challenge. The results presented in this study indicate that the adenovirus vector can be used as a vaccine delivery vector for the development of a PEDV vaccine and is a promising novel vaccine candidate for future prevention and control of porcine epidemic diarrhea (PED), but its efficacy still needs to be improved in the future.

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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

Journal of Virology ◽

10.1128/jvi.01758-18 ◽

2018 ◽

Vol 93 (2) ◽

Cited By ~ 11

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.

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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

Journal of Virology ◽

10.1128/jvi.00406-19 ◽

2019 ◽

Vol 93 (15) ◽

Cited By ~ 10

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.

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Origin, Evolution, and Genotyping of Emergent Porcine Epidemic Diarrhea Virus Strains in the United States

mBio ◽

10.1128/mbio.00737-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 270

Author(s):

Yao-Wei Huang ◽

Allan W. Dickerman ◽

Pablo Piñeyro ◽

Long Li ◽

Li Fang ◽

...

Keyword(s):

United States ◽

Porcine Epidemic Diarrhea Virus ◽

The United States ◽

Genomic Sequences ◽

Evolutionary Divergence ◽

Origin And Evolution ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea ◽

And Control ◽

Complete Genomic

ABSTRACT Coronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U.S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5′-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV. IMPORTANCE The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States.

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

10.22541/au.161460452.27398224/v1 ◽

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.

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The trypsin-enhanced infection of porcine epidemic diarrhea virus is determined by the S2 subunit of the spike glycoprotein

Journal of Virology ◽

10.1128/jvi.02453-20 ◽

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.

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The New Porcine Epidemic Diarrhea Virus Outbreak May Mean That Existing Commercial Vaccines Are Not Enough to Fully Protect Against the Epidemic Strains

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.697839 ◽

2021 ◽

Vol 8 ◽

Author(s):

Qi Gao ◽

Zezhong Zheng ◽

Heng Wang ◽

Songqiang Yi ◽

Guihong Zhang ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Prevention And Control ◽

Clinical Symptoms ◽

Control Measures ◽

Watery Diarrhea ◽

Sequencing Analysis ◽

Diarrhea Virus ◽

Pig Farm ◽

Porcine Epidemic Diarrhea ◽

And Control

Background: On October 30, 2020, piglets and sows in the farrowing house of a pig farm in Jiangxi showed clinical symptoms such as anorexia, watery diarrhea, and vomiting. Epidemiological test, clinical necropsy, and RT-PCR test were carried out on the pig farm for diagnosis. After comprehensive considerations, the disease was judged as porcine epidemic diarrhea virus infection.Results: Thereafter, a series of comprehensive prevention and control measures such as emergency vaccination with autogenous vaccines were adopted. Half a month after inoculation with autogenous vaccines for the farm, the mortality rate of newborn piglets in the farrowing house began to decline, and production gradually returned to being stable. The second-generation sequencing analysis and phylogenetic analysis showed that the porcine epidemic diarrhea virus (PEDV) sequence obtained from the stool and small intestine samples of the diseased pigs on the farm was 97.8% homologous to the vaccine strain. At the same time, antibody testing found that the vaccinated pigs on the pig farm had satisfactory immune response.Conclusion: This case indicated that the PEDV outbreak on the pig farm might aggravate owing to the strain being mutated and could escape the immune protection of the existing vaccine. This case has accumulated technical data for the clinical prevention and control of porcine epidemic diarrhea.

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

Journal of Virology ◽

10.1128/jvi.00227-17 ◽

2017 ◽

Vol 91 (14) ◽

Cited By ~ 34

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.

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