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

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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Porcine Epidemic Diarrhea Virus 3C-Like Protease-Mediated Nucleocapsid Processing: Possible Link to Viral Cell Culture Adaptability

Journal of Virology ◽

10.1128/jvi.01660-16 ◽

2016 ◽

Vol 91 (2) ◽

Cited By ~ 10

Author(s):

Peera Jaru-Ampornpan ◽

Juggragarn Jengarn ◽

Asawin Wanitchang ◽

Anan Jongkaewwattana

Keyword(s):

Cell Culture ◽

Growth Retardation ◽

Porcine Epidemic Diarrhea Virus ◽

Vaccine Development ◽

Wild Type Virus ◽

Economic Losses ◽

Swine Industry ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDVAVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. IMPORTANCE Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.

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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

Veterinary Research ◽

10.1186/s13567-021-00971-5 ◽

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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Isolation and evolutionary analyses of porcine epidemic diarrhea virus in Asia

PeerJ ◽

10.7717/peerj.10114 ◽

2020 ◽

Vol 8 ◽

pp. e10114

Author(s):

Wan Liang ◽

Danna Zhou ◽

Chao Geng ◽

Keli Yang ◽

Zhengying Duan ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Porcine Epidemic Diarrhea Virus ◽

Virus Isolation ◽

Vaccine Development ◽

Evolutionary Analysis ◽

Genetic Characteristics ◽

S Protein ◽

Diarrhea Virus ◽

Comparative Genomics Analysis ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) is a leading cause of diarrhea in pigs worldwide. Virus isolation and genetic evolutionary analysis allow investigations into the prevalence of epidemic strains and provide data for the clinical diagnosis and vaccine development. In this study, we investigated the genetic characteristics of PEDV circulation in Asia through virus isolation and comparative genomics analysis. APEDV strain designated HB2018 was isolated from a pig in a farm experiencing a diarrhea outbreak. The complete genome sequence of HB2018 was 28,138 bp in length. Phylogenetic analysis of HB2018 and 207 PEDVs in Asia showed that most PEDV strains circulating in Asia after 2010 belong to genotype GII, particularly GII-a. The PEDV vaccine strain CV777 belonged to GI, and thus, unmatched genotypes between CV777 and GII-a variants might partially explain incomplete protection by the CV777-derived vaccine against PEDV variants in China. In addition, we found the S protein of variant strains contained numerous mutations compared to the S protein of CV777, and these mutations occurred in the N-terminal domain of the S protein. These mutations may influence the antigenicity, pathogenicity, and neutralization properties of the variant strains.

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

PeerJ ◽

10.7717/peerj.12329 ◽

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.

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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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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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Single-Particle Tracking Porcine Epidemic Diarrhea Virus Moving along Microtubules in Living Cells

Proceedings ◽

10.3390/proceedings2020050124 ◽

2020 ◽

Vol 50 (1) ◽

pp. 124

Author(s):

Yangyang Li ◽

Wei Hou ◽

Jian Wang ◽

Fei Liu

Keyword(s):

Particle Tracking ◽

Single Particle ◽

Porcine Epidemic Diarrhea Virus ◽

Living Cells ◽

Single Particle Tracking ◽

Microtubule Organizing Center ◽

Swine Industry ◽

Diarrhea Virus ◽

Organizing Center ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, has caused severe damage to the swine industry. Although viruses are believed to hijack the microtubule-based transport system, the exact manner of PEDV moving along microtubules has not been fully characterized. In this study, PEDV was labeled with quantum dots which have great brightness and photostability. By using quantum dot-labeled PEDV and single-particle tracking, we were able to systematically dissect the dynamic behaviors of PEDV moving along the microtubules in living cells. We found that PEDVs maintained a restricted motion mode with a relatively stable speed in the cell membrane region while displaying a slow–fast–slow velocity pattern with different motion modes in the cell cytoplasm region and near the microtubule-organizing center. The return movements of small amounts of PEDVs were also observed in living cells. Collectively, our work is crucial for understanding the movement of PEDV in living cells; the proposed work also provides important references for further analysis and studies of the infection mechanism of PEDV.

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Molecular Characterization of Porcine Epidemic Diarrhea Virus and Its New Genetic Classification Based on the Nucleocapsid Gene

Viruses ◽

10.3390/v12080790 ◽

2020 ◽

Vol 12 (8) ◽

pp. 790

Author(s):

Sung-Jae Kim ◽

Van-Giap Nguyen ◽

Thi-My-Le Huynh ◽

Yong-Ho Park ◽

Bong-Kyun Park ◽

...

Keyword(s):

B Cell ◽

Porcine Epidemic Diarrhea Virus ◽

Cell Epitope ◽

N Gene ◽

Swine Industry ◽

Genetic Classification ◽

Nucleocapsid Gene ◽

Diarrhea Virus ◽

B Cell Epitope ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) causes continuous, significant damage to the swine industry worldwide. By RT-PCR-based methods, this study demonstrated the ongoing presence of PEDV in pigs of all ages in Korea at the average detection rate of 9.92%. By the application of Bayesian phylogenetic analysis, it was found that the nucleocapsid (N) gene of PEDV could evolve at similar rates to the spike (S) gene at the order of 10−4 substitutions/site/year. Based on branching patterns of PEDV strains, three main N gene-base genogroups (N1, N2, and N3) and two sub-genogroups (N3a, N3b) were proposed in this study. By analyzing the antigenic index, possible antigenic differences also emerged in both the spike and nucleocapsid proteins between the three genogroups. The antigenic indexes of genogroup N3 strains were significantly lower compared with those of genogroups N1 and N2 strains in the B-cell epitope of the nucleocapsid protein. Similarly, significantly lower antigenic indexes in some parts of the B-cell epitope sequences of the spike protein (COE, S1D, and 2C10) were also identified. PEDV mutants derived from genetic mutations of the S and N genes may cause severe damage to swine farms by evading established host immunities.

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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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