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

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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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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The S Gene Is Necessary but Not Sufficient for the Virulence of Porcine Epidemic Diarrhea Virus Novel Variant Strain BJ2011C

Journal of Virology ◽

10.1128/jvi.00603-18 ◽

2018 ◽

Vol 92 (13) ◽

Cited By ~ 14

Author(s):

Di Wang ◽

Xinna Ge ◽

Dongjie Chen ◽

Jie Li ◽

Yueqi Cai ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Vaccine Development ◽

Economic Losses ◽

Avirulent Strain ◽

Diarrhea Virus ◽

Severe Diarrhea ◽

S Gene ◽

Viral Virulence ◽

Porcine Epidemic Diarrhea ◽

Highly Virulent

ABSTRACT The recently emerged highly virulent variants of porcine epidemic diarrhea virus (PEDV) have caused colossal economic losses to the worldwide swine industry. In this study, we investigated the viral virulence determinants by constructing a series of chimeric mutants between the highly virulent strain BJ2011C and the avirulent strain CHM2013. When tested in the 2-day-old piglet model, wild-type (WT) BJ2011C caused severe diarrhea and death of the piglets within 72 h. In contrast, its chimeric derivative carrying the S gene from CHM2013 (BJ2011C-S CHM ) was avirulent to the piglets. Moreover, reciprocal substitution of the BJ2011C S gene (CHM2013-S BJ ) did not enable CHM2013 to gain any virulence. However, when the whole structural protein-coding region of BJ2011C (CHM2013-SP BJ ) was swapped, CHM2013 started to gain the ability to efficiently colonize the intestinal tract and caused diarrhea in piglets. A further gain of virulence required additional acquisition of the 3′ untranslated region (UTR) of BJ2011C, and the resultant virus (CHM2013-SP + 3UTR BJ ) caused more severe diarrhea and death of piglets. Together, our findings suggest that the virulence of PEDV epidemic strains is a multigenic event and that the S gene is only one of the necessary determinants. IMPORTANCE The recently emerged highly virulent PEDV variants are the major cause of the global porcine epidemic diarrhea (PED) pandemic. The S gene of the variants undergoes remarkable variations and has been thought to be the virulence determinant for the enhanced pathogenesis. Our studies here showed that the S gene is only part of the story and that full virulence requires cooperation from other genes. Our findings provide insight into the pathogenic mechanism of the highly virulent PEDV variants and have implications for future vaccine development.

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Recombinant vesicular stomatitis virus expressing the spike protein of genotype 2b porcine epidemic diarrhea virus: A platform for vaccine development against emerging epidemic isolates

Virology ◽

10.1016/j.virol.2019.05.009 ◽

2019 ◽

Vol 533 ◽

pp. 77-85 ◽

Cited By ~ 6

Author(s):

Yong Ke ◽

Dayi Yu ◽

Fanqing Zhang ◽

Jing Gao ◽

Xiaoyu Wang ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Porcine Epidemic Diarrhea Virus ◽

Vaccine Development ◽

Spike Protein ◽

Diarrhea Virus ◽

Vesicular Stomatitis ◽

Porcine Epidemic Diarrhea

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Piglet immunization with a spike subunit vaccine enhances disease by porcine epidemic diarrhea virus

npj Vaccines ◽

10.1038/s41541-021-00283-x ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Jieshi Yu ◽

Chithra Sreenivasan ◽

Tirth Uprety ◽

Rongyuan Gao ◽

Chen Huang ◽

...

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Neutralizing Antibodies ◽

Subunit Vaccine ◽

Vaccine Development ◽

Spike Protein ◽

Vaccine Research ◽

Disease Symptoms ◽

Diarrhea Virus ◽

Cell Lysate ◽

Porcine Epidemic Diarrhea

AbstractImmunization with an insect cell lysate/baculovirus mixture containing recombinant porcine epidemic diarrhea virus (PEDV) spike protein induced high levels of neutralizing antibodies in both mice and piglets. However, immunization of piglets with this vaccine resulted in enhancement of disease symptoms and virus replication in vaccine recipients exposed to PEDV challenge. Thus, these observations demonstrate a previously unrecognized challenge of PEDV vaccine research, which has important implications for coronavirus vaccine development.

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Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection

The Journal of Immunology ◽

10.4049/jimmunol.2001186 ◽

2021 ◽

pp. ji2001186

Author(s):

Shasha Li ◽

Zixiang Zhu ◽

Fan Yang ◽

Weijun Cao ◽

Jinping Yang ◽

...

Keyword(s):

Membrane Protein ◽

Viral Infection ◽

Porcine Epidemic Diarrhea Virus ◽

Type I ◽

Type I Ifn ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea

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Gene Variations in Cis-Acting Elements between the Taiwan and Prototype Strains of Porcine Epidemic Diarrhea Virus Alter Viral Gene Expression

Genes ◽

10.3390/genes9120591 ◽

2018 ◽

Vol 9 (12) ◽

pp. 591

Author(s):

Tsung-Lin Tsai ◽

Chen-Chang Su ◽

Ching-Chi Hsieh ◽

Chao-Nan Lin ◽

Hui-Wen Chang ◽

...

Keyword(s):

Gene Expression ◽

Porcine Epidemic Diarrhea Virus ◽

The Body ◽

Economic Losses ◽

Viral Gene ◽

Sequence Motif ◽

Cis Acting ◽

Diarrhea Virus ◽

Porcine Epidemic Diarrhea ◽

In Cis

In 2013, the outbreak of porcine epidemic diarrhea (PED) in Taiwan caused serious economic losses. In this study, we examined whether the variations of the cis-acting elements between the porcine epidemic diarrhea virus (PEDV) Taiwan (TW) strain and the prototype strain CV777 alter gene expression. For this aim, we analyzed the variations of the cis-acting elements in the 5’ and 3’ untranslated regions (UTRs) between the PEDV TW, CV777, and other reference strains. We also determined the previously unidentified transcription regulatory sequence (TRS), a sequence motif required for coronavirus transcription, and found that a nucleotide deletion in the TW strain, in comparison with CV777 strain, immediately downstream of the leader core sequence alters the identity between the leader TRS and the body TRS. Functional analyses using coronavirus defective interfering (DI) RNA revealed that such variations in cis-acting elements for the TW strain compared with the CV777 strain have an influence on the efficiency of gene expression. The current data show for the first time the evolution of PEDV in terms of cis-acting elements and their effects on gene expression, and thus may contribute to our understanding of recent PED outbreaks worldwide.

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N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine

Immune Network ◽

10.4110/in.2018.18.e21 ◽

2018 ◽

Vol 18 (3) ◽

Cited By ~ 4

Author(s):

Sae-Hae Kim ◽

Byeol-Hee Cho ◽

Kyung-Yeol Lee ◽

Yong-Suk Jang

Keyword(s):

Porcine Epidemic Diarrhea Virus ◽

Mucosal Vaccine ◽

Spike Protein ◽

Diarrhea Virus ◽

Terminal Domain ◽

Porcine Epidemic Diarrhea ◽

Candidate Molecule

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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 ◽

10.3390/v12010041 ◽

2019 ◽

Vol 12 (1) ◽

pp. 41 ◽

Cited By ~ 2

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.

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A novel biotinylated nanobody-based blocking ELISA for the rapid and sensitive clinical detection of porcine epidemic diarrhea virus

Journal of Nanobiotechnology ◽

10.1186/s12951-019-0531-x ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 4

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.

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