Progress 02/01/15 to 01/31/20
Outputs
Target Audience:Swine farmers, veterinaries, swine vaccine industry, researchers working on veterinary virology, immunology, pathology or swine diseases, virologist Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided opportunities for training three graduate students, one post-doc, one Research Associate, one Research Assistant, and three Visiting Scholars. How have the results been disseminated to communities of interest?The results of our studies have been/will be presented in local, national and international scientific conferences, meetings, and lectures. All results have been.will be published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Highly virulent PEDV, causing up to 100% mortality in neonatal piglets, emerged in China in 2010. It spread to the US in 2013 and killed about 7 million piglets or 10% of the US pig population. PEDV is an economically important enteric pathogen and has caused immense losses among the pork industries. Effective and safe vaccines are urgently needed, but still not available. Traditional live attenuated vaccines (LAVs) based on classical PEDV strains have been used in Asian countries. However, the efficacy of these vaccines is low against the highly virulent PEDV because epidemic PEDV outbreaks continue to occur on routinely vaccinated swine farms. The antigenic differences between the classical and emerging PEDV strains contribute to current vaccine failures. Currently, an inactivated vaccine (Zoetis) and an alphavirus vector-based subunit vaccine based on the highly virulent PEDV strains (Merck) are conditionally licensed in the US. These vaccines are safe, but of questionable efficacy. In this project, we established an infectious clone for a highly virulent PEDV strain PC22A and identified viral genes related to virulence using the infectious clone. A promising PEDV LAV candidate was engineered. The knowledge obtained in this project can be used for the rational design and development of LAVs for PEDV and other emerging animal and human coronaviruses (CoVs), such as porcine deltacoronavirus, swine acute diarrhea syndrome-CoV, MERS-CoV, and SARS-CoV-2. Therefore, the success of this project promotes animal and human health and sustainability of the swine industry. Obj 1. We passaged the tissue culture-adapted (TC) PEDV PC22A strain for 160 passages using two Vero cell lines, Vero-CCL81 and Vero-BI. PC22A-P95 (CCL81) and P100 (BI, no designation subsequently) were subjected to plaque-purification. Two clones of P100 (C4 and C6) were selected and P100C4 was also propagated in Vero-BI cells for an additional 10 passages (P100C4+P10). Eight PC22A-derived viruses [P3, P95C13-(CCL81), P100C4, P100C6, P100C4+P10, P120, P140, and P160] were selected for genomic sequence analysis and virulence studies in neonatal piglets to explore the relatedness between viral mutations and virulence attenuation in pigs. PC22A-P100, P120, P140, and P160 were partially/fully attenuated in neonatal pigs, whereas PC22A-P95C13 (CCL81) and PC22A-P3 remained highly virulent. Sequence analysis revealed that the two virulent viruses had 1, 1, 16 and 2 amino acid (aa) differences in non-structure protein 1 (nsp1), nsp4, spike (S) and membrane (M) proteins, respectively, from the fully attenuated P160. However, the overall pattern of attenuation-related genetic changes in PC22A differed from those of the other four pairs of PEDV wild type strains and their attenuated derivatives in literature. It suggests that PEDV attenuation can occur through multiple molecular mechanisms. Interestingly, the early termination of 9 aa in the S protein of PC22A-P120 and above passages was also found in a mild Chinese field strain FL2013 and a Korean vaccine strain SM98, suggesting that the early termination of S protein may be related to attenuation. This was further investigated using our infectious clone in Obj 3. Obj 2. We studied the pathogenicity and immunogenicity of S INDEL PEDV Iowa106 strain with six conventional sows and their litters. We found that Iowa106 strain was mild in suckling pigs, with 18% mortality depending on factors such as the sow's health and lactation and the piglets' birth weight. Prior infection by Iowa106 and PC21A provided partial (19%) and 100% cross-protection, respectively, to piglets against PC21A challenge. Similarly, we studied the pathogenicity and immunogenicity of PC177-P10 (a 197DEL PEDV at passage level 10) in conventional suckling piglets. PC177-P10 was mild with no piglet mortality. Prior infection by PC177-P10 provided partial cross-protection (12%) to piglets against PC21A challenge. Sequence analyses revealed that the major differences among strains [Iowa106, PC21A, PC22A, and PC177 (P2 and P10)] were mainly in the N-terminal domain (NTD) of the S protein. PC177 had a large 197-aa-deletion in the N-terminal of S protein compared with other PEDV strains. Iowa106 strain had minor insertions (2 aa) and deletions (5 aa) compared with the highly virulent PEDV strains, consistent with its prior designation as an S INDEL strain. PC177-P10 had a 5-nt-deletion in ORF3 compared with PC177-P2, resulting in an early termination of ORF3 protein with 85-aa. Also, PC177-P10 has 9-aa differences from the genome of PC21A in ORF1a (7 aa) and S (2 aa) proteins. These results indicate that the partial attenuation of PC177-P10 was probably the combination of multiple mutations, including the 197DEL in the S protein. Obj 3. Using reverse genetics, we removed ORF3 and replaced this region with a red fluorescent protein (RFP) gene to generate icPEDV-?ORF3-RFP. It replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-?ORF3-RFP-infected pigs were lower than those in PC22A- or icPEDV-infected pigs, suggesting that ORF3 is a minor virulence determinant. Next, we optimized the PEDV PC22A infectious clone (icPC22A) by making the plasmid DNA fragments more stable and improving efficacy of the rescue of recombinant virus from Vero cells compared with the previous infectious clone (icPEDV). Using the new icPC22A platform, we generated the following recombinant PEDVs carrying mutations in S, non-structural protein (nsp)16, and nsp14 proteins and studied virulence of selected recombinant PEDVs in neonatal pigs. We also challenged some pigs to examine protective immunity which is critical for the development of future vaccines. 1) We confirmed that the 197-aa-deletion in the NTD of S is a virulence determinant for PEDV. However, recombinant PEDV lacking the 197-aa did not induce adequate protection in pigs against PC21A challenge. Therefore, the NTD domain of S contains essential immunogenic epitopes and should be retained in LAVs. 2) We studied the role in virulence of the two motifs in the tail of S protein (YEVFEKVHVQ) by generating three recombinant viruses: icPC22A-S2Δ10aa (ΔYEVFEKVHVQ), icPC22A-S2Δ5aa (ΔKVHVQ), and icPC22A-Y1378A (inactivated motif AEVF). icPC22A-S2Δ10aa and icPC22A-Y1378A, but not icPC22A-S2Δ5aa, were partially attenuated in pigs. 3) We verified that the 2'-O methyltransferase domain (2'-O MTase) of nsp16 is a virulence determinant for PEDV in pigs by generating a serial of PEDV mutants, including KDKE4A with quadruple alanine-substitutions in the catalytic tetrad of the 2'-O MTase. 4) We constructed a mutant KDKE4A-SYA by abolishing the "YEAF" endocytosis motif of the S protein of KDKE4A. Compared with icPC22A, KDKE4A and KDKE4A-SYA replicated less efficiently but induced stronger type I and type III interferon responses in vitro. KDKE4A-SYA and KDKE4A were attenuated in pigs. KDKE4A-SYA cross-protected all piglets from virulent PEDV challenge. Our data suggest that KDKE4A-SYA is a good LAV candidate. 5) Coronavirus nsp14 has exoribonuclease activity (ExoN), responsible for proofreading and contributing to replication fidelity. We designed 8 mutants targeting the ExoN catalytic sites, zinc finger or Mg2+-binding site. Only one PEDV mutant was successfully rescued from Vero cells and designed as icPC22A-nsp14-E191A. We attempted to evaluate the pathogenesis of icPC22A-nsp14-E191A in neonatal pigs but failed because no replication of the virus was detected. In vitro, the virus reverted to wild type by passage level 4. So, PEDV mutants carrying mutations at the essential functional sites within nsp14-ExoN domain were either lethal or genetically unstable. The ExoN domain of nsp14 is not an appropriate target for designing a PEDV LAV vaccine.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Yixuan Hou and Qiuhong Wang. 2019. Emerging Highly Virulent Porcine Epidemic Diarrhea Virus: Molecular Mechanisms of Attenuation and Rational Design of Live Attenuated Vaccines. Int J Mol Sci. 2019 Nov 4;20(21). pii: E5478. doi: 10.3390/ijms20215478. (review)
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Hou, Y., H. Ke, J. Kim, D. Yoo, Y. Su, P. Boley, J. Chepngeno, A. N. Vlasova, L. J. Saif, and Q. Wang. 2019. Engineering a live attenuated PEDV vaccine candidate via inactivation of the viral 2'-O methyltransferase and the endocytosis signal of the spike protein. J Virol. 93(15). pii:e00406-19. doi: 10.1128/JVI.00406-19.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Kabita Pandey, Shuhong Zhong, Diego G. Diel, Yixuan Hou, Qiuhong Wang, Eric Nelson, Xiuqing Wang. 2019. GTPase-activating protein-binding protein 1 (G3BP1) plays an antiviral role against porcine epidemic diarrhea virus. Vet Microbiology 236:108392.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Qiuhong Wang, Linda J Saif, Yixuan Hou, Chun-Ming Lin, Thavamathi Annamalai. Molecular attenuation mechanisms of porcine epidemic diarrhea virus (PEDV) in pigs. The 100th Conference for Research Workers in Animal Disease (CRWAD). Poster#102. Chicago, IL. Nov. 2-4, 2019.
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Saif, L.J., Wang Q., Vlasova, A.N., Jung K., Shao, X. Coronaviruses. In: Zimmerman, J. J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W., and Zhang, J. Diseases of Swine. (11th Ed.), John Wiley & Sons, Inc., Hoboken (NJ). 2019.
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Vlasova, A.N., Wang, Q., Jung, K., Langel, S.N., Saif, L.J.2019. Porcine Coronaviruses. In: Recent Advances in Animal Virology, (Malik, Y.S., Singh, R.K., and Yadav, M.P. eds), Springer Natures Text Book, Springer.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Yixuan Hou. 2019. Porcine Epidemic Diarrhea Virus: Molecular Mechanisms of Attenuation and Rational Design of Live Attenuated Vaccines.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2020
Citation:
Xiaoyu Niu, Fanzhi Kong, Yixuan Hou, and Qiuhong Wang. Mutation in the Exoribonuclease of Porcine Epidemic Diarrhea Virus Causes High Genetic Instability. Proc. 38th American Society for Virology Annual meeting, Abstract#?, Fort Collins, Colorado. June 13-17, 2020.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Wang Q, Vlasova AN, Kenney SP, Saif LJ. Emerging and re-emerging coronaviruses in pigs. Curr Opin Virol. 2019 Feb;34:39-49. (Review).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Dongwan Yoo, Yixuan Hou, Hanzhong Ke, Jineui Kim, Yunfang Su, Patricia Boley, Juliet Chepngeno, Anastasia Vlasova, Linda Saif, Quihong Wang. Double-inactivation of nsp16 methyltransferase activity and S protein endocytosis signal increases innate immune response and confers complete protection from PEDV infection. Asian Pig Veterinary Society Congress 2019. Seoul, South Korea, August 25-28.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2020
Citation:
Stephanie N. Langel *, Qiuhong Wang, Anastasia N. Vlasova, Linda J. Saif *. Host factors affecting generation of immunity against porcine epidemic diarrhea virus in pregnant and lactating swine and passive protection of neonates. Pathogens. (Review. Accepted on 02/14/2020)
|
Progress 02/01/18 to 01/31/19
Outputs
Target Audience:Swine farmers, veterinaries, swine vaccine industry, researchers working on veterinary virology, immunology, pathology or swine diseases, virologist Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided opportunities for training three graduate students, one post-doc, one Research Associate, one Research Assistant, and two Visiting Scholars. How have the results been disseminated to communities of interest?The results of our studies have been/will be presented in local, national and international scientific conferences and meetings, then published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?It is the last year for this grant. So we will complete the entire project soon. The senior PhD student is going to graduate in August, 2018. The junior PhD student will get training for future in vitro and in vivo (pig) studies.
Impacts
What was accomplished under these goals?
Highly virulent PEDV, causing up to 100% mortality in neonatal piglets, emerged in China in late 2010. It spread to the US in spring 2013. During the first year of PEDV outbreaks in the US, PEDV killed about 7 million piglets, accounting for 10% pig population. PEDV has become the most economically important porcine enteric viral pathogen and has caused immense economic losses among the pork industries in many countries. Effective and safe vaccines are desperately desired but still not available. Traditional live attenuated vaccines (LAVs) based on classical PEDV strains have been used in Asian countries. However, the efficacy of these vaccines is low against the highly virulent PEDV because epidemic PEDV outbreaks have been reported to occur on routinely vaccinated swine farms in those countries. The high virulence of the emerging PEDV strains and the antigenic differences between the classical and emerging PEDV strains contribute to current vaccine failure. Currently, an inactivated vaccine (Zoetis, Florham Park, NJ) and an alphavirus-based subunit vaccine based on the emerging highly virulent PEDV strains (Merck, Ames, IA) are conditionally licensed in the US. These vaccines are safe, but their efficacy is questionable. Our long term goal is to explore different molecular strategies to generate effective and safe LAVs for PEDV. Firstly, we established an infectious clone for a highly virulent PEDV strain. Then we identified viral genes related to virulence. So the molecular attenuation mechanisms determined in this project can be used for the rational design of LAVs for PEDV and other coronaviruses (CoVs) using the infectious clone platform. Using such broadly applicable strategies, effective LAVs can be produced quickly to respond to the emergence of new PEDV variants and other porcine enteric CoVs, such as porcine deltacoronavirus (PDCoV) and swine acute diarrhea syndrome (SADS)-CoV. Therefore, the success of this project will promote animal health and sustainability of the swine industry. Objs. 1 and 2 had been completed and reported previously. Obj. 3. In previous years, we optimized the infectious clone for PEDV PC22A strain (icPC22A) by making the plasmid DNA fragments more stable and the rescue of recombinant virus from Vero cells with higher efficacy compared with previous version. Using the icPC22A platform, we confirmed that the 197-aa deletion in the N-terminal domain of spike (S) protein is a virulence determinant for PEDV. However, recombinant PEDV lacking the 197-aa did not induce enough protection against the challenge with the highly virulent PEDV. We also determined the two trafficking signaling motifs (YXXF and/or KXHXX) at the end of the S protein and the 2'-O methyltransferase domain (2'-O MTase) of non-structural protein (nsp) 16 are virulence determinants for PEDV in pigs. In 2018, we investigated the molecular attenuation mechanisms of the YXXF and KXHXX mutants (Hou et al., 2019). We generated a recombinant PEDV (KDKE4A-SYA), carrying mutations in both the 2'-O MTase and the YXXF motif, and characterized its replication, pathogenesis and immunogenicity (manuscript in preparation). The KxHxx motif has been reported as the endoplasmic reticulum (ER) retrieval signal, but the function of the YxxF motif in the intracellular sorting of CoV S proteins remains controversial. To determine the roles of the two motifs in the intracellular sorting of S proteins, we constructed 12 plasmids bearing a wild-type (WT) S gene of PC22A or an S gene with different mutations in the two motifs of the cytoplasmic tails. We identified that the YxxF motif of PEDV S protein is an endocytosis signal. We confirmed that the KxHxx motif is an ER retrieval signal for PEDV. The two motifs simultaneously regulate the surface S protein level. Next, we investigated why the deletion of both motifs from the S protein (icΔ10aa), but not the deletion of ER retrieval signal alone (icΔ5aa), significantly attenuates a virulent PEDV. We found that icΔ5aa-infected Vero cells contained significantly higher numbers of mature virions in the Golgi vacuoles than those infected with icΔ10aa. Therefore, icΔ10aa may generate a high level of defective virions (without enough S protein projections on the viral surface), causing attenuation in vivo. We hypothesized that inactivation of both the 2'-O MTase activity of nsp16 and the endocytosis signal of the S protein will significantly attenuate PEDV, but retain its immunogenicity in pigs. Previously, we generated a PEDV mutant KDKE4A with quadruple alanine-substitutions in the catalytic tetrad of the 2'-O MTase of virulent virus icPC22A. Now, we constructed a mutant KDKE4A-SYA by abolishing the endocytosis signal of the S protein of KDKE4A. Compared with icPC22A, KDKE4A and KDKE4A-SYA replicated less efficiently in vitro but induced stronger type I and type III interferon responses. To evaluate the pathogenesis and immunogenicity of these mutants, four groups of 4-day-old gnotobiotic piglets were inoculated orally with 100 PFU/pig of KDKE4A, KDKE4A-SYA, icPC22A, or mock. No mock pigs developed clinical signs. All icPC22A-inoculated pigs had severe diarrhea and died by 6 days post-inoculation (dpi). KDKE4A-SYA and KDKE4A caused milder diarrhea, milder intestinal lesions, less fecal PEDV shedding and lower mortality rates (0% and 16.7% vs. 100%) than icPC22A. At 21 dpi, all surviving pigs were challenged orally with a high dose of icPC22A (6 log10 PFU/pig). No KDKE4A-SYA- and one KDKE4A-inoculated pigs developed diarrhea, whereas all pigs in the mock group had severe diarrhea and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA can be further developed to be an effective and safe PEDV vaccine candidate.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Yunfang Su, Yixuan Hou, Melanie Prarat, Yan Zhang, and Qiuhong Wang. 2018. New variants of porcine epidemic diarrhea virus with large deletions in the spike protein in United States, 2016-2017. Archives of Virology. 163(9):2485-2489 doi: 10.1007/s00705-018-3874-y.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Yixuan Hou, Tea Meulia, Xiang Gao, Linda J Saif, and Qiuhong Wang. 2019. The deletion of Both Tyrosine-Based Endocytosis signal and Endoplasmic Reticulum-Retrieval Signal in the Cytoplasmic Tail of Spike Protein Attenuates PEDV in Pigs. J Virol. 93(2). pii: e01758-18. doi: 10.1128/JVI.01758-18.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Yunfang Su, Yixuan Hou, and Qiuhong Wang. 2019. The enhanced replication of an S-intact PEDV during coinfection with an S1 NTD-del PEDV in piglets. Vet. Microbiol. 228:202-212.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Chun-Ming Lin; Shristi Ghimire; Yixuan Hou; Patricia Boley; Stephanie N. Langel; Anastasia N. Vlasova; Linda J. Saif; Qiuhong Wang 2019. Pathogenicity and immunogenicity of attenuated porcine epidemic diarrhea virus PC22A strain in conventional weaned pigs. BMC Veterinary Research. 15(1):26. doi: 10.1186/s12917-018-1756-x.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Qiuhong Wang, Anastasia N. Vlasova, Scott P. Kenney, Linda J. Saif. 2019. Emerging and re-emerging coronaviruses in pigs. Current Opinion in Virology. 34:39-49. doi: 10.1016/j.coviro.2018.12.001. [Epub ahead of print] Review.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Qiuhong Wang*, Yixuan Hou, and Linda J. Saif. Two trafficking signaling motifs at the end of the spike protein of porcine epidemic diarrhea virus are virulence determinants in pigs. The 25th International Pig Veterinary Society Congress (IPVS 2018) and 2018 International PRRS Symposium, Chongqing, China from June 11 to June 14, 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Yunfang Su, Yixuan Hou, Melanie Prarat, Yan Zhang, and Qiuhong Wang. Archives of Virology. New PEDV variants with a large deletion in the spike protein in United States, 2016-2017. (poster). Proc. 37th American Society for Virology Annual meeting, Abstract#P10-2, College Park, Maryland. July 14-18, 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Yixuan Hou, Yunfang Su, Linda J Saif, and Qiuhong Wang. Porcine Epidemic Diarrhea Virus Lacking Ribose 2O-Methyltransferase Activity is Attenuated in Pigs. (Oral). Proc. 37th American Society for Virology Annual meeting, Abstract#W16-6, College Park, Maryland. July 14-18, 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Qiuhong Wang, Linda J. Saif, Yixuan Hou, Chun-Ming Lin, Xiang Gao, Xinsheng Liu, Thavamathi Annamalai. Molecular attenuation mechanisms of porcine epidemic diarrhea virus (PEDV) in pigs. (oral) Presentation #261. The 99th Conference for Research Workers in Animal Disease (CRWAD). Chicago, IL. December 2-4, 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Yixuan Hou, Tea Meulia, Xiang Gao, Linda J Saif and Qiuhong Wang. A tyrosine-based sorting motif in PEDV spike protein is an endocytosis signal and contributes to viral virulence. (oral) Presentation #260. The 99th Conference for Research Workers in Animal Disease (CRWAD). Chicago, IL. December 2-4, 2018.
|
Progress 02/01/17 to 01/31/18
Outputs
Target Audience:Swine farmers, veterinaries, swine vaccine industry, researchers working on veterinary virology, immunology, pathology or swine diseases, virologist Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities for training one PhD student and one Master student, one post-doc, one new Research Assistant, and one Visiting Scholar. How have the results been disseminated to communities of interest?The results of our studies have been/will be presented in local, national and international scientific conferences and meetings, then published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?It is the no-cost extension year for this grant. So we will complete the entire project by investigating the molecular mechanisms involved in the above two studies of Obj.3 and submitting two papers. We are going to present our research findings at national and international conferences. The PhD student is going to graduate in August, 2018.
Impacts
What was accomplished under these goals?
Emerging highly virulent PEDV was first detected in October 2010 in China. It affected >10 provinces in southern China and caused death of >1,000,000 piglets within one year. During the first year (April 2013-2014) of PED outbreaks in the US, about 7 million piglets died resulting in a $0.9 - $1.8 billion annual loss for U.S. economy. In Japan, it is estimated that > 490,000 pigs died due to PEDV during October 2013 to August 2015. However, an effective vaccine is still lacking. Traditional live, attenuated PEDV vaccines based on classical PEDV strains have been used in Asian countries. However, the efficacy of these vaccines is questionable because epidemic PED outbreaks have been reported to occur on routinely vaccinated swine farms in those countries. The high virulence of the emerging PEDV strains and the differences between the classical and emerging PEDV strains contribute to current vaccine failure. Currently, an inactivated PEDV vaccine (Zoetis, Florham Park, NJ) and an alphavirus-based PEDV subunit vaccine based on the emerging highly virulent PEDV strains (Merck, Ames, IA) are conditionally licensed in the US. These vaccines are safe, but their efficacy is unproven. Our long term goal is to explore different molecular strategies to generate new PEDV vaccines. By establishing an infectious clone for PEDV and identifying regions of the viral genome related to viral virulence, we will have better tools and additional knowledge to develop effective vaccines against PEDV. Since PEDV is so deadly in nursing pigs and can cause major economic losses to the swine industry, the research results will help to develop effective vaccines to prevent and control PED outbreaks, thus promoting animal health and sustainability of the swine industry. In addition, the mechanisms of molecular attenuation determined in this project can be used as broadly applicable strategies for the rational design of attenuated vaccines for coronaviruses (CoVs) using reverse genetics. Therefore, effective vaccines can be produced quickly to respond to the emergence and reemergence of new CoVs. Subsequent to this study, attenuated PEDV variants can be studied further to verify if they are good vaccine candidates. Validated vaccine candidates will be a major achievement for PEDV vaccine development. The established infectious clone of PEDV can be used to study the genetic factors involved in viral virulence and to design effective vaccines based on knowledge obtained for viral determinants of virulence and immunogenicity. Objs. 1 and 2 had been completed and reported previously. Obj. 3. During the study of Obj. 1, we found that a premature terminated (ΔEVFEKVHVQ) spike (S) protein was identified in the 120th and higher passage levels of the cell culture-attenuated original US PEDV strain PC22A. Similar S proteins were also reported for several Vero cell-attenuated PEDV strains or mild field variants. Without affecting any known virus neutralizing epitopes, these PEDV variants lose partial YXXF and/or KXHXX motifs. These two motifs are intracellular trafficking signals critical for retaining S proteins in PEDV assembly sites, the ER-Golgi intermediate compartments. To investigate whether these motifs are virulence determinants, we generated three recombinant viruses with the single or double motif-deletions by introducing stop codons or an amino acid substitution into the infectious clone of virulent PC22A strain (icPC22A): 1) icPC22A-S2Δ10aa (ΔYEVFEKVHVQ); 2) icPC22A-S2Δ5aa (ΔKVHVQ); and 3) icPC22A-Y1378A (inactivated motif AEVF). We orally inoculated (100 PFU/pig) 5-day-old gnotobiotic pigs (n=4-5 per group) with each mutant, and virulent icPC22A. Two pigs were mock inoculated with cell culture medium. Within 52 hours post-inoculation (hpi), all PEDV-infected piglets developed diarrhea. However, piglets inoculated with icPC22A-S2Δ10aa had a significantly lower rate (50%) of severe diarrhea, shed significantly lower titers of infectious virus in feces, and displayed milder intestinal villous atrophy than pigs in the other three virus-inoculated groups. In Vero cells, icPC22A-S2Δ10aa and icPC22A-Y1378A replicated to significantly lower titers but formed significantly larger plaques than icPC22A and icPC22A-S2Δ5aa. Immunofluorescent staining of S proteins of PEDV-infected Vero cells (8 hpi) showed that the amounts of S proteins on the PEDV-infected cell surface were lower for icPC22A and icPC22A-S2Δ5aa than for icPC22A-Y1378A and icPC22A-S2Δ10aa, indicative of the defective in internalization of the S proteins from cell surface of icPC22A-Y1378A and icPC22A-S2Δ10aa. These results suggest that the two motifs at the end of the S protein are virulence determinants of PEDV in pigs. The loss of the motifs likely results in more S proteins on the cell surface, triggering more cell-to-cell membrane fusion to form larger syncytia, but fewer infectious virus particles assembled. Detailed biological functions of these two motifs in PEDV replication are under investigation. Nonstructural protein (nsp) 16 is a highly conserved coronavirus (CoV) 2'-O methyltransferase (MTase) that catalyzes the capping of viral RNAs. We hypothesized that the inactivation of nsp16 will induce host innate anti-viral responses, leading to PEDV attenuation in pigs. Using the infectious cDNA clone icPC22A, we generated five PEDV mutants by introducing amino acid (aa)-substitutions into key residues of nsp16: mutants with catalytic tetrad "KDKE" substituted by a single alanine (D129A) or quadruple alanine (KDKE4A); SAM-binding site mutant (D98A); nsp10/nsp16 interface mutant (R85AR86A); and putative Mg2+-binding site mutant (T57E). All mutants were rescued in Vero cells. The mutant KDKE was selected to study pathogenicity and immunogenicity in pigs because it was more sensitive to IFN-I responses and replicated less efficiently than the other four mutants in vitro. Three groups of 4-day-old gnotobiotic piglets were inoculated orally with 100 PFU/pig of the KDKE mutant, the virulent icPC22A, or mock. No mock pigs developed clinical signs. The KDKE4A mutant caused milder diarrhea, lower mortality rates (16.7%, 1/6 vs. 100%, 8/8), less fecal viral RNA shedding and milder intestinal lesions than virulent icPC22A. We challenged all KDKE4A and mock surviving pigs with icPC22A (106 PFU/pig) at 22 days post-inoculation. No pigs in the KDKE4A group developed diarrhea, whereas all pigs in the mock group had severe diarrhea. Thus the KDKE mutant is attenuated in pigs and retained immunogenicity, and may be a candidate PEDV vaccine. We will further characterize the nsp16 mutants by quantifying the MTase activity and induction of type III IFN responses in infected porcine intestinal cells, and confirming the stability in vitro.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2017
Citation:
Wang, Q., Feng, L., Saif, L.J. Chapter 68: Porcine epidemic diarrhea. In: Koos Coetzer (Chief Editor), Infectious Diseases of Livestock (http://demo.anipedia.org/)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Yixuan Hou, Chun-Ming Lin, Masaru Yokoyama, Boyd L. Yount, Douglas Marthaler, Arianna L. Douglas, Shristi Ghimire, Yibin Qin, Ralph S. Baric, Linda J. Saif, and Qiuhong Wang. Deletion of a 197 amino acid-region in the N-terminal domain of spike protein attenuates PEDV. XIVth International Nidovirus Symposium. June 4th - 9th, 2017 in Kansas City, Missouri, USA.(Poster#S6P-7)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Wang, Q.; Hou, Y.; Saif, L.J. (2017). Two trafficking signaling motifs at the end of the spike protein of porcine epidemic diarrhea virus are virulence determinants in pigs. CRWAD 2017, December 3-5, 2017. Chicago, IL. [Abstract#157]
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Hou Y, Lin CM, Yokoyama M, Yount BL, Marthaler D, Douglas AL, Ghimire S, Qin Y, Baric RS, Saif LJ*, and Wang Q*. 2017. Deletion of a 197 amino acid-region in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus. J Virol. 91(14). pii: e00227-17.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Chun-Ming Lin, Yixuan Hou, Douglas G. Marthaler, Xiang Gao, Xinsheng Liu, Lanlan Zheng, Linda J. Saif*, Qiuhong Wang*. 2017. Attenuation of an original US porcine epidemic diarrhea virus strain PC22A via serial cell culture passage. Veterinary Microbiology 201: 6271.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Annamalai T, Lin CM, Gao X, Liu X, Lu Z, Saif LJ*, Wang Q*. 2017. Cross protective immune responses in nursing piglets infected with a US spike-insertion deletion porcine epidemic diarrhea virus strain and challenged with an original US PEDV strain. Vet Res. 48(1):61.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Yunfang Su, Yixuan Hou, Melanie Prarat, Yan Zhang, and Qiuhong Wang. New variants of porcine epidemic diarrhea virus with large deletions in the spike protein in the United States, 2016-2017. Archives of Virology. (Submitted)
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Ghimire, Shristi. 2017. Screening for enteric coronaviruses in fecal samples of feral pigs of California, USA. MS Thesis. The Ohio State University.
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Progress 02/01/16 to 01/31/17
Outputs
Target Audience:Swine farmers, veterinaries, swine vaccine industry, researchers working on veterinary virology, immunology, pathology or swine diseases Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities for training one new Research Assistant, one post-doc, one PhD student and one Master student. How have the results been disseminated to communities of interest?The results of Obj. 1 have been published as one peer-reviewed research paper. The 2nd manuscript describing results of partial Obj. 2 has been submitted to Journal of Virology. Also, a review paper, a book chapter, and a White paper on PEDV targeting different audience were written. The results of our studies have been/will be presented in local, national and international scientific conferences and meetings. What do you plan to do during the next reporting period to accomplish the goals?Obj. 3. We will use the established reverse genetics system to engineer recombinant PEDV mutants to identify other genes related to virulence, immunogenicity and recombination.
Impacts
What was accomplished under these goals?
Emerging highly virulent PEDV was first detected in October 2010 in China. It affected >10 provinces in southern China and caused death of >1,000,000 piglets within one year. During the first year (April 2013-2014) of PED outbreaks in the US, about 7 million piglets died resulting in a $9 million to $1.8 billion annual loss for U.S. economy. In Japan, it is estimated that > 490,000 pigs died due to PEDV during October 2013 to August 2015. However, an effective vaccine is still lacking. Traditional live, attenuated PEDV vaccines based on classical PEDV strains have been used in Asian countries. However, the effectiveness of these vaccines is questionable because epidemic PED outbreaks have been reported to occur on routinely vaccinated swine farms in China. The high virulence of the emerging PEDV strains and/or the differences between the classical and emerging PEDV strains contribute to vaccine failure. Currently, an inactivated PEDV vaccine (Zoetis, Florham Park, NJ) and an alphavirus-based PEDV subunit vaccine based on the emerging highly virulent PEDV strains (Merck, Ames, IA) are conditionally licensed in the US. These vaccines are safe, but their effectiveness is unproven. Our long term goal is to explore different molecular strategies to generate new PEDV vaccines. By establishing an infectious clone for PEDV and identifying regions of the viral genome related to viral virulence, we will have better tools and additional knowledge to develop effective vaccines against PEDV. Since PEDV is so deadly in nursing pigs and can cause major economic losses to the swine industry, the research results will help to develop effective vaccines to prevent and control PED outbreaks, thus promoting animal health and sustainability of the swine industry. In addition, the mechanisms of molecular attenuation determined in this project can be used as broadly applicable strategies for the rational design of attenuated vaccines for coronaviruses (CoVs) using reverse genetics. Therefore, effective vaccines can be produced quickly to respond to the emergence and reemergence of new CoVs. Subsequent to this study, attenuated PEDV variants can be studied further to verify if they are good vaccine candidates. Validated vaccine candidates will be a major achievement for PEDV vaccine development. The established infectious clone of PEDV can be used to study the genetic factors involved in viral virulence and to design effective vaccines based on knowledge obtained for viral determinants of virulence and immunogenicity. Obj. 1. Sequence analysis revealed that the virulent viruses [P3 and P95C13 (CCL81)] had one, one, sixteen (including an early termination of nine amino acids) and two amino acid differences in non-structure protein 1 (nsp1), nsp4, spike and membrane proteins, respectively, from the fully attenuated P160. However, the overall pattern of attenuation-related genetic changes in PC22A differed from those of the other four pairs of PEDV wild type strains and their attenuated derivatives. These results suggest that PEDV attenuation can occur through multiple molecular mechanisms. The results have been published as one research paper in Veterinary Microbiology (Lin et al., 2016). Obj. 2. We orally inoculated neonatal, conventional suckling piglets with TC-PC177 or the highly virulent PC21A to compare the pathogenicity. Within 7 days post-inoculation, 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 of TC-PC177 can induce cross-protection against highly virulent PEDV strain challenge, all the surviving piglets were challenged with PC21A at 3 weeks post-inoculation. 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. Obj. 3.The optimized infectious clone for the highly virulent PC22A strain can be used to steadily generate PEDV variants carrying different mutations to study gene functions. 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.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Chun-Ming Lin, Yixuan Hou, Douglas G. Marthaler, Xiang Gao, Xinsheng Liu, Lanlan Zheng, Linda J. Saif, Qiuhong Wang. 2016. Attenuation of an original US porcine epidemic diarrhea virus strain PC22A via serial cell culture passage. Veterinary Microbiology 201 (2017) 6271.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Chun-Ming Lin, Linda J. Saif, Douglas Marthaler and Qiuhong Wang. 2016. Evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains. Virus research. 226:20-39
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Yixuan Hou, Chun-Ming Lin, Masaru Yokoyama, Boyd L. Yount, Douglas Marthaler, Arianna L. Douglas, Shristi Ghimire, Yibin Qin, Ralph S. Baric, Linda J. Saif, and Qiuhong Wang. Deletion of a 197 amino acid-region in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets. (submitted to Journal of Virology)
- Type:
Book Chapters
Status:
Submitted
Year Published:
2017
Citation:
Saif, L.J., Wang Q., Vlasova, A.N., Jung K., Shao, X. Coronaviruses, In: Zimmerman, J. J., Zhang, J. (11th Ed.), Diseases of Swine. Wiley-Blackwell, Ames, IA.
- Type:
Other
Status:
Published
Year Published:
2016
Citation:
Nov. 2016, The PI (Qiuhong Wang) and co-PI (Linda J. Saif) were invited by National Pork Producers Council and wrote a White paper entitled "Potential Risk of Porcine Epidemic Diarrhea Virus (PEDv) from legally imported pork from the United States".
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Yixuan Hou, Chun-Ming Lin, Xiang Gao, Zhongyan Lu, Xinsheng Liu, Yibin Qin, Linda J. Saif and Qiuhong Wang. Evaluation of the virulence of a porcine epidemic diarrhea virus with a 197 amino acid-deletion in the spike protein. Proc. 35th American Society for Virology Annual meeting, W35-11, Blacksburg, Virginia. June 18-22, 2016.
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Progress 02/01/15 to 01/31/16
Outputs
Target Audience:Swine farmers, veterinaries, swine vaccine industry, researchers working on veterinary virology, immunology, pathology or swine diseases Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities for training one post-doc, two PhD student and one Master student. How have the results been disseminated to communities of interest?A portion of results of objectives 2 and 3 were published as three peer-reviewed publications. One and two manuscripts describing results of objectives 1 and 2, respectively, are currently in preparation. Also, the results of our studies have been/will be presented in local, national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Obj. 1. PEDV PC22A at high pass level showed lower virulence and can be used to generate high titer virus stocks, which potentially can be used to develop cost-effective inactivated and/or live attenuated vaccines to protect pigs from current highly virulent PEDV in the field. Therefore, we plan to conduct the second stage of animal study, higher dose of viral inoculation in conventional piglets, to exam whether PC22A-P120 (BI) and PC22A-P160 (BI) are completely attenuated and able to induce protective immunity in sows to protect their piglets from the original US PEDV infection. Obj. 2. We are working on the mucosal and serum immune responses induced by the original US PEDV PC21A and S-INDEL Iowa106 and their cross-reactivities. Evaluation of the pathogenicity and immunity of another PEDV variant, S 197DEL PC177, is currently ongoing. Obj. 3. Based on the results of Obj. 1, we will use the established reverse genetics system to engineer PEDV mutants to identify genes related to virulence and recombination.
Impacts
What was accomplished under these goals?
Obj. 1. PC22A strain was continuously passaged in Vero-CCL81 cells to passage level 95 [P95 (CCL81)] and in Vero-BI cells to passage level 160 [P160 (BI)]. In vitro studies found that from P65 (CCL81 and BI), the viral infectious titers increased 3 log10 to titers of 8 log10 PFU/ml, indicating better adaptation of the virus to cell culture. In vivo CDCD piglet studies showed that PC22A-P100 (BI) and PC22A-P120 (BI) had 0% mortality, whereas PC22A-P95 (CCL81) and PC22A-P3 had 100% mortality. Although PC22A-P100 (BI) and PC22A-P120 (BI) still caused diarrhea in some piglets, those pigs had delayed virus shedding, decreased and delayed onset of clinical signs, milder intestinal lesions and lower viral antigen scores. Genomic sequence analysis revealed 17 and one amino acid differences between virulent PC22A-P95 (CCL81) and attenuated PC22A-P120 (BI) in S and M proteins, respectively. These amino acid changes between virulent and attenuated PC22A differed from those in the other 3 pairs of PEDV [83P5 (P1 vs P100), DR13 (virulent vs attenuated), and YN (P1 vs P144)]. One 2 amino acid (aa)-deletion (DEL) in S1 of PC22A-P120 (BI) also exists in the virulent US PEDV strain Minnesota188, suggesting that the 2 aa-DEL is not directly related to attenuation. However, an early termination of 9 aa in the S protein of PC22A-P120 (BI) was also found in a reportedly mild Chinese strain FL2013 and Korean vaccine strain SM98, suggesting that the early termination of S protein is probably related to attenuation. Obj. 2. All PEDV S-INDEL Iowa106- and original US PEDV PC21A-inoculated piglets developed diarrhea. However, the severity of clinical signs, mortality (0-75%) and fecal PEDV RNA shedding titers varied among the four S-INDEL Iowa106-inoculated litters. Compared with the original PC21A, piglets euthanized/died acutely from S-INDEL Iowa106 infection had relatively milder villous atrophy, lower antigen scores and more limited intestinal infection. Two of four S-INDEL Iowa106-infected sows and the original PC21A-infected sow showed anorexia and watery diarrhea for 1-4 days. After original PC21A challenge, a subset (13/16) of S-INDEL Iowa106-inoculated piglets developed diarrhea, whereas no pigs in the original PC21A-inoculated pigs had diarrhea. Our results suggest that the virulence of S-INDEL PEDV Iowa106 was less than the original US PEDV PC21A in suckling pigs, with 100% morbidity and 18% (6/33) overall (0 to 75%) mortality in suckling pigs depending on factors such as the sow's health and lactation and the piglets' birth weight. Prior infection by S-INDEL Iowa106 provided partial cross-protection to piglets against original PC21A challenge at 21-29 DPI. Objective 3. The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. Importantly, recombinant PEDV was rapidly transmitted to the un-inoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. Also, icPEDV-ΔORF3-RFP replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-ΔORF3-RFP-infected pigs were lower than those in wild type virus- or icPEDV-infected pigs, suggesting decreased virulence in vivo.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
1. Liu X., Lin C.M., Annamalai T., Gao X., Lu Z., Esseili M.A., Jung K., El-Tholoth M., Saif L.J., Wang Q. 2015. Determination of the infectious titer and virulence of an original US porcine epidemic diarrhea virus PC22A strain. Vet Res. 46:109.
2. Lin C.M., Annamalai T., Liu X., Gao X., Lu Z., El-Tholoth M., Hu H., Saif L.J., Wang Q. 2015. Experimental infection of a US spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original US PEDV infection. Vet Res. 20:134.
3. Beall, A., B. Yount, C. M. Lin, Y. Hou, Q. Wang, L. Saif and R. Baric (2016). Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A. MBio 7(1).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
1. Xinsheng Liu, Chun-Ming Lin, Annamalai Thavamathi, Xiang Gao, Zhongyan Lu, Malak A. Esseili, Kwonil Jung, Linda J. Saif, Qiuhong Wang. Determination of the infectious titer and virulence of US original porcine epidemic diarrhea virus (PEDV) strain PC22A. 2015 OARDC Annual Conference. Columbus, OH, USA. April 16, 2015.
2. Chun-Ming Lin, Susan E. Sommer-Wagner, Xinsheng Liu, Xiang Gao, Zhongyan Lu, Mohamed S Eltholoth, Yixuan Hou, Linda J. Saif and Qiuhong Wang. Generation of attenuated US PEDV vaccine candidates via continuous cell culture passages. 2015 OARDC Annual Conference. Columbus, OH, USA. April 16, 2015.
3. Wang, Q., Lin, C.M., Annamalai, T., Liu, X., Lu, Z., Gao, X., Hu, H., and Saif, L.J. 2015. Evaluation of the virulence of a US spike-insertion deletion (S-INDEL) PEDV and its cross-protection against the US original highly virulent PEDV in suckling piglets. 7th international symposium on emerging and re-emerging pig diseases. Kyoto, Japan. June 21-24.
4. Lin, C.M., Sommer-Wagner, S., Marthaler, D., Hou, Y., Gao, X., Liu, X., Liu, Z., Eltholoth, M. S., Saif, L.J. and Wang, Q. 2015. Attenuation of US original porcine epidemic diarrhea virus strain PC22A via continuous cell culture passages. Conference of Research Workers in Animal Diseases. Chicago, USA. Dec 7-9.
5. Q. Wang. Molecular attenuation mechanisms of Porcine Epidemic Diarrhea Virus in pigs. 2015 USDA-NIFA AFRI Project Director Workshop (Animal Health, Animal Well-Being, and Food Security, etc.). Chicago, IL. December 3, 2015.
6. Q. Wang, Combating enteric viruses to improve swine and human health. 2015 NC1202 Regional Research Technical Committee, Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety. Chicago, IL. December 5-6, 2015.
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