Passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein

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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Identification and characterization of a novel linear epitope in the spike protein of the porcine epidemic diarrhea virus

Acta Virologica ◽

10.4149/av_2019_111 ◽

2019 ◽

Vol 63 (01) ◽

pp. 88-95

Author(s):

L. GONG ◽

Y. GONG ◽

Y. LIN ◽

J. QIN ◽

Y. LIU ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Spike Protein ◽

Linear Epitope ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea ◽

Identification And Characterization

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The 3.1-Angstrom Cryo-electron Microscopy Structure of the Porcine Epidemic Diarrhea Virus Spike Protein in the Prefusion Conformation

Journal of Virology ◽

10.1128/jvi.00923-19 ◽

2019 ◽

Vol 93 (23) ◽

Cited By ~ 21

Author(s):

Daniel Wrapp ◽

Jason S. McLellan

Keyword(s):

Receptor Binding ◽

Porcine Epidemic Diarrhea Virus ◽

Binding Domain ◽

Host Cells ◽

Spike Protein ◽

Diarrhea Virus ◽

Putative Receptor ◽

Cryo Electron Microscopy ◽

Sialic Acid Binding ◽

Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus that has a significant agricultural and economic impact due to the high mortality rate associated with infection of neonatal piglets. Like other coronaviruses, PEDV makes use of a large, trimeric spike (S) glycoprotein to mediate membrane fusion and gain entry into host cells. Despite the importance of the spike protein in viral entry and host immune responses, high-resolution structural information concerning this large macromolecular machine has been difficult to obtain. Here, we report the cryo-electron microscopy structure of the PEDV S protein in the prefusion conformation at a resolution of 3.1 Å. Our studies revealed that the sialic acid-binding domain at the N terminus of the S1 subunit has an orientation that is substantially different from that observed in the previously determined spike structure from human alphacoronavirus NL63. We also observed dissociated S1 subunit trimers wherein the putative receptor-binding domains exist in a conformation differing from that observed in the intact spike proteins, suggesting that the PEDV receptor-binding domain undergoes conformational rearrangements akin to those that have been described in the related betacoronaviruses. Collectively, these data provide new insights into the biological processes that mediate alphacoronavirus attachment, receptor engagement, and fusion triggering while also identifying a source of conformational heterogeneity that could be manipulated to improve PEDV vaccine antigens. IMPORTANCE Coronavirus spike proteins are large, densely glycosylated macromolecular machines that mediate receptor binding and membrane fusion to facilitate entry into host cells. This report describes the atomic-resolution structure of the spike protein from porcine epidemic diarrhea virus, a pathogenic alphacoronavirus that causes severe agricultural damage. The structure reveals a novel position for the sialic acid-binding attachment domain in the intact spike. We also observed shed fusion-suppressive capping subunits that displayed the putative receptor-binding domain in an accessible conformation. These observations provide a basis for understanding the molecular mechanisms that drive the earliest stages of alphacoronavirus infection and will inform future efforts to rationally design vaccines.

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Production of soluble truncated spike protein of porcine epidemic diarrhea virus from inclusion bodies of Escherichia coli through refolding

Protein Expression and Purification ◽

10.1016/j.pep.2016.05.018 ◽

2016 ◽

Vol 126 ◽

pp. 77-83 ◽

Cited By ~ 4

Author(s):

Da-Chuan Piao ◽

Yoon-Seok Lee ◽

Jin-Duck Bok ◽

Chong-Su Cho ◽

Zhong-Shan Hong ◽

...

Keyword(s):

Escherichia Coli ◽

Porcine Epidemic Diarrhea Virus ◽

Inclusion Bodies ◽

Spike Protein ◽

Diarrhea Virus ◽

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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