Progress 06/15/19 to 06/14/24
Outputs
Target Audience:Swine farmers, veterinarians, swine vaccine industry, researchers working on virology, immunology, pathology, and/or swine diseases Changes/Problems:We encountered technical, personnel, and financial difficulties, partially due to COVID-19 pandemic, but we tried our best and completed Objectives 1 and 2. Objective 3 is not performed. Technically, it was more difficult than we expected to engineer a recombination-resistant PEDV genome for vaccine development. This is partially due to the unique genomic characteristics of PEDV: PEDV has slightly different TRS-core sequences (TRS-CSs) for different ORFs and the body TRS-CSs often overlap the end of the upstream ORF. This made rewiring the TRS-CSs extremely difficult; it took us 2.5 years to complete it. Also, the entire project was much delayed due to COVID-19 pandemic. We encountered difficulty in personnel in the past four years: Our experienced lab manager decided to retire in October 2020 after working eight years in our lab. We hired a new lab manager, but he left after 1 month. Since 2021, it had been difficult to recruit a qualified Research Assistant. Finally, the budget became very tight. During the CoVID-19 shut-down and half-time working period, we still needed to pay the graduate students and research assistant 100% salary, benefits, and stipend. Our university/department also raised their salary and stipend due to the high inflation rates in recent years. The high inflation rates also made experimental reagents and materials more expensive. In 2021, I attempted to request COVID supplemental fund from USDA, but was told that "NIFA did not receive supplemental funds to deal with COVID related setbacks. So we don't have funds available to assist in this capacity, especially given how many researchers likely suffered setbacks during the initial COVID period". In such a situation, I used our departmental funding for my technician to support current PhD student and completed Objectives 1 and 2. We do not have any resources to perform Objective 3. What opportunities for training and professional development has the project provided?This project has provided opportunities for training 2 PhD students (Xiaoyu Niu became an Assistant Professor at North Carolina A&T State University in August 2023; Mingde Liu is going to graduate in Autumn 2024), one undergraduate student (Jacqueline Anne Spieles who completed the 1st year at College of Wooster), one Research Associate (Xiaohong Susan Wang), and one Visiting Scholar (Fanzhi Kong). 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, and published in peer-reviewed journals and book chapters. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Porcine epidemic diarrhea virus (PEDV) is a deadly alphacoronavirus for piglets, causing porcine epidemic diarrhea (PED) with high mortality rates of 50-100%. Safe and effective PEDV vaccines are urgently needed but are not available. Due to the ability to induce broad and prolonged protective immunity and the convenient administration routes, live attenuated vaccines (LAVs) are promising arms for controlling PED. However, conventional LAVs are unsafe because they either revert to virulent strains or recombine with wildtype virus to generate new virulent viruses when they are applied in the field. Using the state-of-the art reverse genetics technology, we developed LAV candidates that are resistant to revert to virulence or recombine with wildtype PEDV. Therefore, the application of such LAVs at farms is safe. These discoveries have a significant impact for pig health and the sustainability of swine industry. Also, PEDV infection of pigs is a good model to test our universal strategies for the generation of recombination-resistant LAVs for coronaviruses (CoVs). The success of this project will aid in innovative vaccine design against PEDV and newly emerging animal and human CoVs. Objectives 1 and 2 have been completed and the major accomplishments are listed: Identification of attenuating mutations in PEDV nonstructural protein 1 (nsp1). Coronavirus nsp1 inhibits cellular gene expression and antagonizes interferon (IFN) response. We hypothesized that a recombinant PEDV carrying mutations at the conserved residues N93 and N95 of nsp1 induces higher IFN responses and is more sensitive to IFN responses, leading to virus attenuation. We mutated PEDV nsp1 N93 and N95 to A93 and A95 to generate the recombinant N93/95A virus using the infectious clone of a highly virulent PEDV strain, PC22A (icPC22A), and evaluated N93/95A virus in vitro and in vivo. Compared with virulent PEDV icPC22A, the N93/95A mutant replicated to significantly lower infectious titers, triggered stronger type I and III IFN responses, and was more sensitive to IFN treatment in vitro. In 5-day-old gnotobiotic piglets, N93/95A caused significant lower mortality rate of 25% (1/4) than icPC22A (100% mortality rate), and protected all survival pigs from virulent PEDV challenge, indicating that it was partially attenuated and retained viral immunogenicity. In summary, nsp1 N93A and N95A can be targets for the development of LAVs for PEDV. Because CoV nsp1 is conserved among alphacoronaviruses (alpha-CoVs) and betacoronaviruses (beta-CoVs), it may be a good target for vaccine development for other alpha-CoVs or beta-CoVs. Generation of a recombination-resistant PEDV genome by engineering the transcriptional regulatory sequences (TRSs) for LAV development. The TRSs are critical in regulating CoV discontinuous transcription in virus replication. We hypothesize that recoding the TRS core sequences (TRS-CSs) of PEDV can make the recombination impossible between the engineered vaccine virus and wild-type viruses because the recombinant virus cannot transcribe sub-genomic messenger RNAs (sgmRNA) and produce progeny infectious viruses due to the incompatibility between the recoded and wildtype TRS-CSs. We used an infectious clone-derived reporter PEDV, dORF3-enhanced green fluorescent protein (EGFP), as the backbone to generate a remodeled TRS (RMT) mutant that carries the recoded leader and body TRS-CSs (except for the EGFP). The RMT and dORF3-EGFP showed comparable replication efficiency in Vero cells. However, the incompatibility between the rewired and wild-type TRS-CSs led to few EGFP in RMT-infected cells. Also, RMT and dORF3-EGFP had a similar attenuated phenotype, replication efficiency, and protective immunogenicity in neonatal pigs. RNA sequencing analysis indicated that EGFP transcription directed by the heterogeneous TRS-CSs was significantly reduced to an extremely low level. Meanwhile, recombinant viruses were not detected in Vero cells and in pigs that were co-infected with RMT and a PEDV S-INDEL strain, Iowa106. In vitro and in vivo passaging of the RMT did not result in reversion mutations in the rewired TRS-CSs, introduced gaps, and disrupted wild-type TRSs. In summary, the RMT mutant was resistant to recombination and genetically stable and can be further optimized (e.g., deletion of the EGFP) to serve as a platform to develop safe PEDV LAVs. Generation of an optimized recombination-resistant PEDV genome RMTv1. In the RMT mutant, an unexpected 189-nt-insertion containing highly repetitive sequences in front of E gene and one "G"-deletion within the N gene TRS-CS were detected. Also, the RMT contains an exogenous EGFP gene in replace with PEDV's accessory gene ORF3. All these should be corrected before the RMT can be a "ready-to-go" chassis for LAV development. We stepwise deleted the 189-nt-insertion, inserted the "G", and removed the EGFP gene to rescue three viruses: 1) RMT-ΔG with the correction for the 189-nt-insertion, 2) RMT-CID (corrected insertion and deletion) with both corrections for the 189-nt-insertion and the "G"-Del, and 3) RMT-v1 with the removal of the EGFP gene from RMT-CID. The recombinant viruses were plaque-purified, and their genome sequences were confirmed. In vitro viral growth kinetics showed that RMT-ΔG, RMT-CID, and RMT-v1 had similar replication efficiency but lower than RMT. Because the 189-nt-insertion in front of the E gene is the only difference between viruses RMT and RMT-ΔG, it suggested the potential enhancement function of the 189-nt-insertion for viral replication. In the future, the PEDV E gene can be used as a model to precisely regulate coronavirus sgmRNA transcription by modifying the insertion size in the TRS region. This mechanism has the potential to enhance other immunogenic protein expression, ultimately improving the protective efficacy and decreasing the production cost of LAVs. These findings highlight the importance of fine-tuning genetic alterations in vaccine development to strike a balance between attenuation and vaccine efficacy. Engineering six PEDV LAV candidates based on RMTv1, and the selection of one candidate for future vaccine studies in sows and their newborn suckling piglets. We hypothesized that with multiple distant attenuation mutations in different viral genes and the recombination-resistant platform, these LAV candidates will have a lower chance to revert to a virulent strain via mutations or recombination. We generated 6 PEDV mutants by adding individual or the combinations of two inactive mutations of nsp1, nsp15, and nsp16 into the RMT-v1 backbone: 1) RMT-v1-nsp1, 2) RMT-v1-nsp15, 3) RMT-v1-nsp16, 4) RMT-v1-nsp1+nsp15, 5) RMT-v1-nsp1+nsp16, and 6) RMT-v1-nsp15+nsp16. We examined the growth kinetics and genetic stability of these LAVs in vitro. Finally, we tested the pathogenesis and immunogenicity of RMT-v1 and one selected mutant (RMT-v1-nsp1+nsp15) in neonatal gnotobiotic pigs. We found that RMTv1-nsp1+15 did not cause severe diarrhea or death and protected all the pigs from mortality post challenge. These results indicate that RMTv1-nsp1+15 was attenuated and retained good immunogenicity and can be further tested in pregnant sows and their newborn suckling piglets in the future. RMTv1 caused death in 40% (2/5) of piglets, significantly lower than the 100% mortality rate caused by virulent PEDV icPC22A, indicating partial attenuation. Both RMTv1 and RMTv1-nsp1+15 can be LAV chassis for quick updating vaccines against novel PEDV strains by introducing desired attenuating mutations or directly replacing the S gene with that of an emerging PEDV strain. Objective 3: Due to the technical, personnel, and financial difficulty encountered in the past years (see Changes/Problems section), we cannot perform this objective.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Mingde Liu, Xiaoyu Niu, Qiuhong Wang. Optimize the PEDV recombination-resistant platform for safe and effective live attenuated vaccine development. Oral. The Conference of Research Workers in Animal Diseases (CRWAD) Annual meeting. January 20-23, 2024. Chicago, IL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Mingde Liu, Xiaoyu Niu, Shaomin Yang, Jelmer W. Poelstra, Hua Zhu, and Qiuhong Wang. Development of a recombination-resistant live attenuated vaccine for PEDV. (Poster and 3-min flash talk). Abstract#W20-17. Proc. 43rd American Society for Virology Annual meeting, June 24-28, 2024. Columbus, OH.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Niu, X., Liu, M., Yang, S., Xu, J., Hou, Y.J., Liu, D., Tang, Q., Zhu, H., Wang, Q., 2023, A recombination-resistant genome for live attenuated and stable PEDV vaccines by engineering the transcriptional regulatory sequences. Journal of virology, 97(12):e0119323.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Qiuhong Wang. Developing recombination-resistant attenuated vaccines to fight porcine epidemic diarrhea virus (PEDV). International Porcine Reproductive and Respiratory Syndrome Symposium (IPRRSS 2024), Emerging Viruses session. August 7-9, 2024. Yantai, China.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Xiaoyu Niu. 2022. Study towards the development of effective and safe live attenuated PEDV vaccines. Doctor of Philosophy, Ohio State University, Comparative and Veterinary Medicine.
|
Progress 06/15/22 to 06/14/23
Outputs
Target Audience:Swine farmers, veterinaries, swine vaccine industry, researchers working on virology, immunology, pathology, and/or swine diseases Changes/Problems:It was more difficult than we expected to engineer a recombination-resistant PEDV platform. This was partially due to the unique genomic characteristics of PEDV: PEDV has slightly different TRS-core sequences (TRS-CSs) and the body TRS-CSs often overlap the end of the upstream. This made rewiring the TRS-CSs extremely difficult. Also, the entire project was much delayed because we encountered difficulty in personnel in the past three years: the 1st PhD student graduated in August 2022 after four years study. Our lab manager retired in October 2020. We hired a new lab manager, but he left after 1 month. During 2020-2021, it had been difficult to recruit a qualified Research Assistant. In August 2022, the first PhD student who was mainly working on this project graduated and left our lab for a post-doctoral position. The 2nd PhD student, who was recruited in August 2019, left suddenly in December 2021. This generated a gap between the 1st student and the 3rd PhD student, who started his PhD program in August 2020 but was less experienced in reverse genetics. In this reporting period, the 3rd PhD student has been trained to master the techniques and has started to make significant progress. Finally, the budget became very tight. During the CoVID-19 shut-down and half-time working period, we still needed to pay the graduate students and research assistant 100% salary, benefits, and stipend. We also raised their salary and stipend due to the high inflation rates in recent years according to the university's new policy. The high inflation rates also made experimental reagents and materials more expensive. From 2022, we do not have funds to support a research assistant. In such a situation, we will attempt to complete Aim 2 in the final year but cannot do Aim 3. The team has worked hard to obtain further funding to continue our excellent research. What opportunities for training and professional development has the project provided?This project has provided opportunities for training one 2.5 PhD students: Xiaoyu Niu became an Assistant Professor in the U.S. in August 2023; Jiayu Xu is a PhD student at the Columbus campus of The Ohio State University; Mingde Liu is a 3rd Yr student. 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. We also communicated with Animal Health sector of a pharmaceutical company for potential collaboration on the sow studies of the vaccine candidate. What do you plan to do during the next reporting period to accomplish the goals?We plan to optimize the recombination-resistant RMT-v1 platform and generate PEDV vaccine candidates by adding multiple attenuation mutations, especially targeting the innate immunity antagonist genes, and test their pathogenesis and immunogenicity in neonatal piglets.
Impacts
What was accomplished under these goals?
Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), which is characterized by diarrhea, vomiting, dehydration, and weight loss. It emerged in the US in 2013, causing the death of 10% of US pig population and $0.9 to $1.8 billion in economic losses within one year. To control and prevent PED, USDA conditionally licensed two vaccines for use in sows in 2014: 1) The viral vector-based vaccine expressing the PEDV S protein (Merck), 2) the inactivated vaccine based on a highly virulent PEDV isolate (Zoetis). However, both vaccines showed limited efficiency when they were used to immunize PEDV-naive sows. Although it is more difficult to develop safe and effective live attenuated vaccines (LAVs) than other types, it is still worthy to develop them because LAVs mimic natural virus infection in hosts, but replicate weakly and do not cause disease, and can induce both humoral and cellular protective immunity. Our long-term goal is to develop safe and efficacious LAVs for PEDV using reverse genetics technology and the knowledge obtained from previous studies about the molecular mechanisms of coronavirus (CoV) replication. Also, PEDV infection of pigs is a good model to test our universal strategies for the generation of reversion- and recombination-resistant LAV candidates for CoVs. The success of this project will aid in innovative vaccine design against PEDV and newly emerging animal and human CoVs. Objective 1. Previously, our lab generated a recombination-resistant PEDV mutant RMT. RMT carries the recoded leader and body transcriptional regulatory sequences (TRSs) that are incompatible with the wildtype TRSs. So, a recombinant virus between the RMT and field PEDV strains cannot complete the transcription of sub-genomic RNAs, leading to no expression of viral structural proteins to produce infectious viruses. However, when we performed RNA-sequencing to analyze the RNA profiles of the RMT mutant, we found that the subgenomic RNA (sgRNA) of E gene was the most abundant sgRNA population. This was strange because we expected that the sgRNA of N gene was the most abundant among all sgRNAs, as the wildtype PEDV. Extensive sequence analyses revealed an unexpected 189-nt-insertion containing highly repetitive sequences in front of E gene and one "G" deletion within the N gene TRS of the RMT. Therefore, these unexpected mutations of RMT needed to be corrected. Also, the RMT contains an enhanced green fluorescent protein (EGFP) gene, which should be absent in LAVs. Using the infectious clone of RMT, we deleted the 189-nt-insertion and inserted a "G" into the plasmid using NEBuilder HiFi DNA assembly master mix and Q5 site-directed mutagenesis kit (NEB, Ipswich, MA). The mutant virus RMT-v1 was rescued from Vero cells. The RMT-v1 mutant replicated similarly to the RMT mutant with a peak titer of 6 log10 50% tissue culture infectious dose (TCID50)/mL at 60 hours post-inoculation (hpi). In summary, we corrected the unexpected insertion and deletion of RMT and generated RMT-v1. Currently, we are deleting EGFP from RMT-v1 to generate RMT-v2. We hope that the RMT-v2 is a ready-to-use recombination-resistant PEDV platform. Our work is a breakthrough towards the completion of our final goal that is to generate safe and effective PEDV LAVs.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Xiaoyu Niu, Mingde Liu, Jiayu Xu, Qiuhong Wang. Development of one recombination-resistant platform for PEDV live attenuated vaccine by remodeling the transcription regulation sequences. (submitted to JVI)
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
Qiuhong Wang. (2022). Isolation of porcine epidemic diarrhea virus from clinical samples. In: Wang, L. (eds) Animal Coronaviruses (2nd Ed.), Springer Protocols Handbooks. Humana, New York, NY. DOI : 10.1007/978-1-0716-2091-5
- Type:
Book Chapters
Status:
Submitted
Year Published:
2024
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. (12th Ed.), John Wiley & Sons, Inc., Hoboken (NJ).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Qiuhong Wang, Xiaoyu Niu, Mingde Liu, Shaomin Yang, Jiayu Xu, Yixuan Hou, Dongxiao Liu, Qiyi Tang, Hua Zhu. A recombination-resistant PEDV genome for the development of live attenuated vaccines by targeting the transcriptional regulatory sequences. Flash talk and P21. The XVIth International Nidovirus Symposium. May 14th - 18th, 2023 at Montreux, Switzerland.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Xiaoyu Niu, Jiayu Xu, Mingde Liu, and Qiuhong Wang. (Oral) Engineering a recombination resistant PEDV by targeting the TRS for live attenuated vaccine development. The 103 Conference for Research Workers in Animal Disease (CRWAD). Chicago, IL. Jan. 21-24, 2023 (attended virtually).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Evolution of porcine epidemic diarrheic virus and prevention and control strategies in pig farms. Session 5 Disease detection and decontamination. The 7th NAU Swine Conference & 2023 Zhongshan Swine Expo. June 30, 2023. Nanjing, China.
|
Progress 06/15/21 to 06/14/22
Outputs
Target Audience:Swine farmers, veterinaries, swine vaccine industry, researchers working on veterinary virology, immunology, pathology and/or swine diseases, virologist Changes/Problems:Due to the COVID-19 pandemic, our research progress has been much delayed due to 1) CoVID-19-related policy, and 2) the difficulty to recruit graduate students with a good background. During the CoVID-19 shut-down and half time working period, we still needed to pay the employee 100% salary, benefits, tuitionand stipend. Additionally,high inflation rates in recent years also made it hard to complete the proposed study with the original budget. What opportunities for training and professional development has the project provided?This project has provided opportunities for training one senior PhD student and junior PhD student. 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?We plan to generate PEDV vaccine candidates by adding multiple attenuation mutations, especially targeting the innate immunity antagonist genes and test them in pigs.
Impacts
What was accomplished under these goals?
Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), which is characterized by diarrhea, vomiting, dehydration, and weight loss. It emerged in the US in 2013, causing the death of 10% of US pig population and $0.9 to $1.8 billion in economic losses within one year. To control and prevent PED, USDA conditionally licensed two vaccines for use in sows in 2014: 1) The viral vector-based vaccine expressing the PEDV S protein (Merck), 2) the inactivated vaccine based on a highly virulent PEDV isolate (Zoetis). However, both vaccines showed limited efficiency at farms. Although it is more difficult to develop safe and effective live attenuated vaccines (LAVs) than other types, it is still worthy to develop them because LAVs mimic natural virus infection in hosts, but replicate weakly and do not cause disease, and can induce both humoral and cellular protective immunity. Our long-term goal is to develop safe and efficacious LAVs for PEDV using reverse genetics technology and the knowledge obtained from previous studies about the molecular mechanisms of coronavirus (CoV) replication. Also, PEDV infection of pigs is a good model to test our universal strategies for the generation of reversion- and recombination-resistant LAV candidates for CoVs. The success of this project will aid in innovative vaccine design against PEDV and newly emerging animal and human CoVs. Objective 1. The generation of one recombination resistant PEDV infectious clone for LAV development by recoding the transcription regulation sequences (TRSs). During CoV replication, a discontinuous transcription mechanism is employed to generate a set of sub-genomic RNA intermediates. TRSs are critical elements regulating the process. We hypothesized that a recoded TRS core sequence that is incompatible with wildtype (WT) TRSs can block recombination events between the engineered virus carrying the recoded TRS and field strains carrying WT TRSs because a recombinant carrying incompatible TRSs is nonviable. This may serve as a strategy to design safe LAVs. Our objective is to engineer such a recombination resistant PEDV by remodeling the TRS. First, we generated a reporter PEDV strain dORF3-EGFP, whose ORF3 is replaced by enhanced green fluorescent protein (EGFP), based on the infectious clone of a highly virulent PEDV strain PC22A. Then we designed primer sets to identify the putative TRS-CSs and RdRp template switch sites for dORF3-EGFP. Finally, using the dORF3-EGFP as a backbone, we designed one remodeled TRS (RMT) mutant that carries the recoded leader and body TRSs, except the one for the EGFP gene. Multi-step growth curves showed comparable replication efficiency between the RMT mutant and dORF3-EGFP virus in Vero cells. No/extremely low EGFP level in the RMT-infected cells indicated the incompatibility between the recoded and WT TRSs. Experimental infection of neonatal gnotobiotic (Gn) pigs showed that the RMT mutant had similar attenuated phenotype and replication efficiency to dORF3-EGFP. Challenge of the pigs with the highly virulent parental PEDV strain icPC22A at 19 days later showed that both RMT and dORF3-EGFP protected the pigs from severe diarrhea and death, indicating that they retained good immunogenicity. The modified TRSs were genetically stable after passaging in vitro and in vivo. We also co-infected the cells/pigs with RMT (or dORF3-EGFP as a positive control) and a PEDV S-INDEL strain Iowa106 to test whether the RMT blocks recombination. We only found potential recombinants from the pigs co-infected with dORF3-EGFP and Iowa106, but not the group co-infected with RMT and Iowa106. These results indicated that RMT mutant is resistant to recombination due to incompatibility between the recoded and WT TRSs and genetically stable. In previous years, we have tried different strategies but failed to generate one recombination resistant PEDV infectious clone. This is because PEDV has inconsistent TRS-CSs and the body TRS-CSs often overlap the end of the upstream ORFs within the genome, making the modification of the TRS regions challenging. In this successful story, we rewired the TRSs by introducing silent mutations to disrupt the original TRS-CSs and artificial gaps between the overlapping TRS-CSs and ORFs. In summary, the recombination resistant RMT can serve as a platform for LAV development by adding additional desired mutations along its genome.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Xiaoyu Niu, Mingde Liu, Jiayu Xu, Qiuhong Wang. Generation of one recombination resistant PEDV infectious clone for live attenuated vaccine development by recoding the transcription regulation sequences. Proc. 41st American Society for Virology Annual meeting, July 16-23, 2022. Madison, WI.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Xiaoyu Niu, Fanzhi Kong, Jiayu Xu, Mingde Liu, Qiuhong Wang. 2022. Mutations in porcine epidemic diarrhea virus nsp1 cause increased viral sensitivity to host interferon responses and attenuation in vivo. J Virol. 96:e0046922.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Xiaoyu Niu and Qiuhong Wang. 2022. Prevention and Control of Porcine Epidemic Diarrhea: The Development of Recombination-Resistant Live Attenuated Vaccines. Viruses. 14:1317. doi.org/10.3390/v14061317
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Xiaoyu Niu, Fanzhi Kong, Jiayu Xu, Mingde Liu, and Qiuhong Wang. Mutations in PEDV nsp1 causes increased viral sensitivity to host interferon responses and attenuation in vivo. The 102 Conference for Research Workers in Animal Disease (CRWAD). Abstract # 179 (Oral) Chicago, IL. Dec. 5-7, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Xiaoyu Niu, Mingde Liu, Jiayu Xu, Qiuhong Wang. Generation of one recombination resistant PEDV infectious clone for live attenuated vaccine development by recoding the transcription regulation sequences. 2022 OSU CFAES Annual Conference. Wooster, OH, USA. April 11, 2022.
|
Progress 06/15/20 to 06/14/21
Outputs
Target Audience:Swine farmers, veterinaries, swine vaccine industry, researchers working on veterinary virology, immunology, pathology and/or swine diseases, virologist Changes/Problems:Since the mutation in the nsp14 exoribonuclease of PEDV is highly instable, we will replace nsp14 with nsp1 as one of the targets for the design of LAVs for this project. Due to COVID-19 pandemic, our research project has been delayed due to CoVID-19-related policy at Ohio State University: 1) non-CoVID-19-related research cannot be performed during March - June 2020 and 50% facility occupancy policy from July 2021 to June 2021. What opportunities for training and professional development has the project provided?This project has provided opportunities for training one graduate student, one Research Associate, 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?We plan to generate PEDV vaccine candidates containing multiple targeted attenuation mutations. We will continue to explore alternative ways to make PEDV resistant to recombination.
Impacts
What was accomplished under these goals?
Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), which is characterized by diarrhea, vomiting, dehydration, and weight loss. It emerged in the US in 2013, causing the death of 10% of US pig population and $0.9 to $1.8 billion in economic losses within one year. To control and prevent PED, USDA conditionally licensed two vaccines for use in sows in 2014: 1) The viral vector-based vaccine expressing the PEDV S protein (Merck), 2) the inactivated vaccine based on a highly virulent PEDV isolate (Zoetis). However, both vaccines showed limited efficiency at farms. Although it is more difficult to develop safe and effective live attenuated vaccines (LAVs) than other types, it is still worthy to develop them because LAVs mimic natural virus infection in hosts, but replicate weakly and do not cause disease, and can induce both humoral and cellular protective immunity. Our long-term goal is to develop safe and efficacious LAVs for PEDV using reverse genetics technology and the knowledge obtained from previous studies about the molecular mechanisms of coronavirus (CoV) replication. Also, PEDV infection of pigs is a good model to test our universal strategies for the generation of reversion- and recombination-resistant LAV candidates for CoVs. The success of this project will aid in innovative vaccine design against PEDV and other emerging animal and human CoVs. Objective 1. In previous year, we reported the design and rescue of one rPEDV icPC22A-nsp1-N93/95A (N93/95A) mutant carrying nsp1 mutations N93A and N95A. This year, we sequenced the genome of the rescued N93/95A mutant and confirmed that it contains the designed but no other mutations. Next, we characterized its phenotype in vitro and in vivo. In both Vero and LLC-PK1 cells, N93/95A mutant replicated to 0.5 - 1.0 log10 TCID50-lower titers compared with the parental virus icPC22A. A significantly higher ratio (100-fold increase) of genomic RNA titer:infectious virus titer were tested in the N93/95A-infected cells compared with icPC22A-infected cells at 24 hours post-inoculation (hpi), suggesting that N93/95A replicated in a very low efficiency at the early stage of infection. Since the nsp1 protein of PEDV was reported as a major antagonist to host interferon (IFN) responses, we analyzed the replication of N93/95A mutant in Vero cells pretreated with IFNs. The N93/95A was more sensitive to type I IFNβ-pretreatment than icPC22A at a concentration of 200 unit/10^5 cells. As for type III IFNλs, N93/95A showed increased sensitivity to IFNλ1 of less than 25 ng/mL and to IFNλ3 of less than 50 ng/mL. To evaluate whether N93/95A mutant triggers enhanced IFN responses in vitro, we infected the IFN-competent LLC-PK1 cells with icPC22A or the N93/95A mutant and quantified the mRNA levels of swine IFNβ, IFNλ1, and IFNλ3. Compared with icPC22A, N93/95A mutant induced significantly higher mRNA levels of all the three IFNs at 24 hpi. Collectively, these results suggested that N93/95A mutant exhibited significantly reduced replication and higher sensitivity to host IFN responses in vitro. Finally, we investigated the pathogenesis and immunogenicity of the N93/95A mutant in neonatal gnotobiotic (Gn) pigs. All icPC22A-inoculated pigs (100%, 5/5) developed severe diarrhea from 1 day post-inoculation (dpi) and died within 6 dpi (100%). In comparison, all N93/95A-inoculated piglets had severe diarrhea, but only one of the four pigs died at 5 dpi (25% mortality rate). The N93/95A-infected piglets shed significantly lower and delayed peak infectious viral titers [4.80 ± 0.76 log10 TCID50/mL at 2 dpi] in feces than the icPC22A-inoculated pigs [5.80 ± 0.33 log10 TCID50/mL at 1 dpi]. Histopathological examination showed that N93/95A mutant induced milder lesions than icPC22A. To examine the immunogenicity, all the remaining pigs were challenged with a high dose of icPC22A at 22 dpi. No mortality was observed in the N93/95A group, whereas one mock-challenged pigs died at 8 days post-challenge (dpc). By 9 dpc, pigs in the N93/95A group had mild diarrhea for 3.67 ± 0.58 days but not severe diarrhea. However, all the mock-challenged pigs (100%, 4/4) developed severe diarrhea for 6.00 ± 0.82 days. Moderate level of virus neutralizing antibody titers in the sera were detected in the N93/95A-inoculated pigs at 22 dpi/0. In summary, the N93A and N95A mutations of nsp1 attenuated PEDV but retained viral immunogenicity. So, they can be the targets for the design of PEDV LAVs. Because nsp1 is exclusively found in α-CoVs and β-CoVs and the N93 and N95 residues are conserved among the nsp1 proteins, these two amino acid residues may be potential targets for the vaccine development for other α-CoVs and β-CoVs.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Xiaoyu Niu, Fanzhi Kong, Yixuan J. Hou, and Qiuhong Wang. 2021. Crucial Mutation in the Exoribonuclease Domain of nsp14 of PEDV Leads to High Genetic Instability During Viral Replication. Cell & Bioscience. 11. DOI: 10.1186/s13578-021-00598-1
- Type:
Conference Papers and Presentations
Status:
Published
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. 39th American Society for Virology Annual meeting, (planned Fort Collins, Colorado) June 13-17, 2020. (Virtual meeting)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Qiuhong Wang, Xiaoyu Niu, and Fanzhi Kong. Porcine epidemic diarrhea virus nsp1 is an interferon antagonist and a determinant of virulence. The 101st Conference for Research Workers in Animal Disease (CRWAD). Abt# 564. Dec. 5-8, 2020. (Virtual meeting)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Xiaoyu Niu, Fanzhi Kong, Yixuan Hou, and Qiuhong Wang. Abt# 563. Mutation in the Exoribonuclease of Porcine Epidemic Diarrhea Virus Causes High Genetic Instability. The 101st Conference for Research Workers in Animal Disease (CRWAD). Dec. 5-8, 2020. (Virtual meeting)
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Progress 06/15/19 to 06/14/20
Outputs
Target Audience:Swine farmers, veterinaries, swine vaccine industry, researchers working on veterinary virology, immunology, pathology or swine diseases, virologist Changes/Problems:Due to COVID-19 pandemic, our research project has been delayed for at least 2 months since the start of emergency status of campus in the middle of March. What opportunities for training and professional development has the project provided?This project has provided opportunities for training two graduate students, one undergraduate, one Research Associate, 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?We plan to complete Obj. 1.
Impacts
What was accomplished under these goals?
Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), which is characterized by diarrhea, vomiting, dehydration and weight loss. Although all ages of pigs are susceptible to PEDV, neonatal piglets are most vulnerable to it with 100% morbidity and up to 100% mortality. Since 2010, highly virulent PEDV have attacked the swine industry of many countries. PEDV emerged in the US in 2013, causing the death of 10% of US pig population and $0.9 to $1.8 billion in economic losses within one year. To control and prevent PED, US Department of Agriculture conditionally licensed two vaccines for use in sows in 2014: 1) The viral vector-based vaccine expressing the PEDV S protein (Merck), and 2) the inactivated vaccine based on a highly virulent PEDV isolate (Zoetis). However, both vaccines showed limited efficiency at farms. Although it is more difficult to develop safe and effective live attenuated vaccines (LAVs) than the other types, it is still worthy to develop them because LAVs mimic natural virus infection in hosts, but replicate weakly and do not cause disease, and can induce both humoral and cellular protective immunity. Our long-term goal is to develop safe and efficacious LAVs for PEDV using reverse genetics technology and the knowledge obtained from previous studies about the molecular mechanisms of PEDV replication, attenuation, and coronavirus recombination. Also, PEDV infection of pigs is a good model to test our universal strategies for the generation of reversion- and recombination-resistant LAV candidates for coronaviruses (CoVs). The success of this project will aid in innovative vaccine design against PEDV and other emerging animal and human CoVs. Obj 1. 1) Recovery of PEDV nsp1 mutant. Upon PEDV infection, the interferon (IFN) antiviral pathway of pigs is activated to suppress viral replication. PEDV express several proteins that act as IFN antagonists. Nonstructural protein 1 (nsp1) is one of them. In this study, we mutated PEDV nsp1 to reduce its anti-IFN function. We designed three recombinant PEDV mutants carrying mutations in the critical sites of nsp1 using the infectious clone of a highly virulent PEDV strain PC22A (icPC22A): 1) icPC22A-nsp1-T23A, 2) icPC22A-nsp1-N93/95A, and 3) icPC22A-nsp1-Δ93-95. PCR based mutagenesis was employed to introduce these mutations. By sanger sequencing, we observed several unintended mutations in the plasmids responsible for the T23A and Δ93-95 mutations, but not in the N93/95A mutation. After electroporating Vero cells with the full-length mRNA genome of icPC22A-nsp1-N93/95A, infectious virus was rescued. icPC22A-nsp1-N93/95A was subjected to plaque purification. Clone #4 of icPC22A-nsp1-N93/95A showed obvious cytopathic effect (CPE) and was picked up. After passaging once in Vero cells, viral RNA was extracted. Reverse-transcription (RT)-PCR was performed to amplify overlapping genomic fragments. RT-PCR products were purified and are ready for the whole genome sequencing to determine the genome integrity of the recombinant virus. 2) Rewiring transcription regulatory sequence (TRS) responsible for PEDV recombination. A safe LAV should be resistant to recombination with circulating virulent PEDV strains because recombination may result in the generation of new virulent PEDV variants. During CoV replication, a set of sub-genomic (sg) RNA intermediates, which is indispensable for CoV viability, are generated. All these sgRNAs start from different open reading frames (ORFs) of genomic RNA but all share identical 3'-end and a short sequence in the 5'-end, called the leader sequence (~90 nt). In this sequence, there is a leader transcription regulatory sequence (TRS) that is identical or highly homologous to the different body TRSs located in front of each ORF in the genome. We used different strategies and constructed fragments or plasmids carrying rewired TRS for PEDV. The infectious clone of icPC22A-ΔORF3-GFP, whose accessory protein ORF3 was replaced by green fluorescent protein (GFP), was used as the backbone. 2.1 We designed a rewired TRS carrying silent mutations. So, the mutant virus contains the same amino acid sequence to icPC22A. The rewired TRS (CUCGAU) for GFP transcription had two mutations compared with the wild type TRS (CUAGAC) counterpart. We constructed a set of DNA fragments using overlap extending PCR. The DNA fragments, under the control of T7 promoter, contains either rewired TRS (CUCGAU) or wild type TRS (CUAGAC) followed by the coding sequences for GFP, PEDV envelop (E) protein, membrane (M) protein and nucleocapsid (N) proteins. The transcription of GFP was controlled by the mutated TRS. After sequence confirmation, the fragments were subjected to in vitro transcription for mRNA synthesis. The mRNA bearing rewired or wild type TRSs were used to transfect Vero cells that was infected by icPC22A, serving as the helper virus. The mRNA with either rewired TRS or wild type TRS were transfected into Vero cells infected by the helper virus, icPC22A. Cells transfected with mRNAs with either rewired TRS or wild type TRS showed green fluorescence and identical profile for sgRNAs, suggesting rewired TRS (CUCGAU) with two mutations is still compatible with wild type TRS. So, we cannot use this rewired TRS for the generation of LAVs. 2.2 Unlike the strategy used in 2.1, two patterns of rewiring TRSs leading to conservative amino acid replacement, in which an amino acid in a protein is replaced by another amino acid with similar biochemical properties, were designed based on the infectious cDNA clone, icPC22A-ΔORF3-GFP. Pattern 1 (UUAAGU) and pattern 2 (UUAGUA) of rewiring TRS bears 3 and 4 mutations, respectively, compared with the wild type leader TRS (CUAAAC). The TRS responsible for GFP transcription in the plasmid for icPC22A-ΔORF3-GFP was replaced by the pattern 1 of rewiring TRS by PCR based mutagenesis. The plasmid bearing pattern 1 mutated TRS was successfully constructed. We will perform sequence confirmation followed by the generation of recombinant viruses. Upon the achievement of this, we can construct recombinant PEDV mutants carrying a GFP controlled by rewired TRS that enables us to evaluate the compatibility between the rewired TRS and wild type TRS of PEDV. A rewired TRS that cannot compatible with the wild type TRS can be used to design recombination resistant LAVs for PEDV.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Qiuhong Wang. Rational design of attenuated vaccines for porcine epidemic diarrhea virus. The 100th Conference for Research Workers in Animal Disease (CRWAD). Poster #180. Chicago, IL. Nov. 2-4, 2019.
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