Source: TENNESSEE STATE UNIVERSITY submitted to NRP
ANTIVIRAL POTENCY AND FUNCTIONAL NOVELTY OF PORCINE INTERFERON-OMEGA SUBTYPE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1015513
Grant No.
2018-67016-28313
Cumulative Award Amt.
$277,000.00
Proposal No.
2017-05786
Multistate No.
(N/A)
Project Start Date
Jul 1, 2018
Project End Date
Jun 30, 2022
Grant Year
2018
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Dept. Agri. & Environ. Sci.
Non Technical Summary
Innate immune interferons (IFNs), particularly type I IFNs, are primary mediators regulating antiviral immunity. These antiviral cytokines have evolved remarkable molecular and functional diversity to confront ever-evolving viral threats. Pigs have the largest and an expanding type I IFN family consisting of nearly 60 functional genes that encode seven IFN subtypes including multigene subtypes of IFN-alpha and omega. Whereas subtypes such as IFN-alpha and beta have been widely studied, the unconventional IFN-omega subtype has barely been investigated. We have evolutionarily defined the porcine IFN family, and preliminarily showed that porcine IFN-omega subtype has evolved several novel features including, (1) a signature multi-gene subtype expanding particularly in bats and ungulates, (2) emerging isoforms that have much higher antiviral potency than typical IFN-alpha, (3) cross-species high antiviral (but little anti-proliferative) activity in cells of humans and other mammalian species, and (4) potential action through non-canonical signaling pathways. Our proposal is focused on antiviral potency of porcine IFN-omegas investigating their evolutionary and functional diversity, signaling specificity, and optimization of novel antivirals against devastating viral diseases. Three Aims will address this goal: (1) determine the expression and inflammatory regulation of porcine IFN-omega subtype in the lung and lung cells during the viral infections; (2) determine their antiviral potency and signalig novelty exerted by porcine IFN-omega subtype, and (3) develop IFN-omega-based biosimilars for antiviral therapeutics. This project will, for the first time in an animal species, establish state-of-the-art procedures for efficient characterization of the molecular and functional spectrums of unconventional IFNs, which will further IFN-based novel antiviral design.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3114030109050%
3043510108050%
Knowledge Area
311 - Animal Diseases; 304 - Animal Genome;

Subject Of Investigation
3510 - Swine, live animal; 4030 - Viruses;

Field Of Science
1090 - Immunology; 1080 - Genetics;
Goals / Objectives
The expansion of IFN-ω molecular diversity in pigs represents a signature event of type I IFN evolution in mammalian species. We hypothesize that this subtype expansion confers functional diversification that is necessary in regulating immune responses against species-specific and even cross-species viral infections. Focusing on the IFN-ω members and their antiviral and inflammatory regulation, our goal is to family-wide characterize porcine innate immune IFNs for their therapeutic potential and functional spectrum, which will be profiled against two RNA viruses (PRRSV and SIV) that have high impact on swine and potentially human health. Three Specific Aims will be functionally integrated to achieve our goal: (1) determine the expression and inflammatory regulation of porcine IFN-omega subtype in the lung and lung cells during the viral infections; (2) determine their antiviral potency and signalig novelty exerted by porcine IFN-omega subtype, and (3) develop IFN-omega-based biosimilars for antiviral therapeutics. This project will, for the first time in an animal species, establish state-of-the-art procedures for efficient characterization of the molecular and functional spectrums of unconventional IFNs, which will further IFN-based novel antiviral design.
Project Methods
Experimental Approach: We have used SYBR® Green-based RT-PCR assays to determine ~30 IFN transcripts with some cross-subtype non-specificity (Fig. 3) [7,8], which will be improved by using a TaqMan® probe-based procedure [78]. We have also established reliable IFN bioassays [7,17] and will use these to analyze the activity of IFN peptides. This will enable us to identify the subset of IFNs that are secreted in response to viral infection in vitro and in vivo and will be used to develop relevant 'omics' approaches if needed. A newly developed SmartFlare™ probe technology (EMD Millipore Corporation, Billerica, MA), will be used to dynamically detect mRNA of key IFNs in live cells. Therefore, our procedures for analysis of IFN expression will use several approaches to reciprocally confirm each other at the gene, RNA and potentially protein levels. For inflammatory regulation, we will treat lung tissue or cell cultures with IFN peptides, and consequent inflammatory responses will be examined per pathological examination and inflammatory cytokine detection. An animal test will be implemented in Aim 3 after promising IFN candidates are identified through in vitro analysisUsing the panel of our expressed or isolated IFN peptides [7,8,20], we will focus on antiviral potency against PRRSV and SIV in porcine cells, particularly airway epithelial, macrophage, and dendritic cells [18,23]. These cells represent cell lineages directly targeted by either or both viruses and are critical for antiviral regulation [18,22,23]. Porcine IFN-ω1 and, especially IFN-ω5, exerted cross-species and stronger antiviral potency than IFN-α/β and differently induced the expression of typical ISGs. We will examine the novel features of porcine IFN-ω signaling including: (1) extracellular and intracellular interaction/dependence with receptor subunits, IFNAR1 and IFNAR2 [97,98]; (2) different dependence of STAT1-3 and the phosphorylation of these transcription factors [4-6]; (3) difference in induction of a series of ISG genes

Progress 07/01/18 to 06/30/22

Outputs
Target Audience:Group I: Industry and academic professionals including swine immunologists, virologists, nutritionists, consultants and other researcher workers, as well as producers, veterinarians and veterinary researchers. Efforts: 1) Conference presentation at scientific meetings such as the Annual Conference of The American Association of Immunologists (AAI), International PRRS Symposium, and the Conference of Research Workers in Animal Diseases (CRWAD), International Nidovirus Symposium (Note: Although our abstracts have been accepted, several conferences were cancelled due to COVID19 crisis); 2) TSU's day at the capital city of Tennessee.; 3) publications in book chapters, and peer-reviewed and open-assessed scientific journals including Frontier Immunology, Genes, Heliyon, Pathogens, Research in Veterinary Science, Viruses, Vaccine, 4) Cross-campus professional seminars in the national and international academic institutes (mostly online) Group II: Graduate and undergraduate students. Efforts: 1) In addition to the first effort above, data/discoveries have also been presented specifically at the University annual Research Day/Forum; and 2) other in-campus and inter-campus student research forums; 3) exchange and publication of students' research theses; 4) incorporation and introduction in lectures/labs of animal health/disease/immunology courses; 5) the department and inter-department seminar presentation, topic lectures or lab research training Group III: Other types of public audience. Efforts: Through the activity of TSU agricultural extension system, other local news media and online broadcasting (YouTube, Laboratory and the PI's Institutional Website etc.) Changes/Problems:Two terms of non-cost extension was appliedand approved for efficient use of the remaining budget, training students and finalizing results/data for publication and dissemination. Otherwise, no major changes/problems are needed or have met except some unexpected delay due to COVID-19 shut-downs. What opportunities for training and professional development has the project provided?Three undergraduate research assistants: Ms. Kendall McCoy: Biological Sci Major, TLSAMP Scholarship Student. Joined the project in June, 2020, submitted an abstract to TLSAMP Conference and co-authored a relevant paper in the Vaccines Journal Ms. Autumn Peterson: Biological Sci Major, TLSAMP Scholarship Student. Joined the project in June, 2020, submitted an abstract to TLSAMP Conference and co-authored a relevant paper in the Vaccines Journal Ms. LeAnn Lopez: Animal Sci Major, Dean Scholarship Student. Joined the project in April, 2020, submitted an abstract to TLSAMP Conference, co-authored a relevant paper in the Vaccines Journal and co-authored a relevant paper in the Viruses Journal Four graduate students: Mr. Jordan Jennings: Joined this project from January to September 2020, and successfully graduated from Food and Animal Science Master Program with five papers published in Frontier Immunology, Cells, Biomolecules, Viruses; he was immediately hired after graduation as Research Scientist at Vanderbilt University Medical School Ms. Yun Tian: Partially joined this project during this entire reporting period, submitted an abstract to 2020 CORWAD Conference for student research award competition and co-authored four relevant papers in Hellion, Pathogens and Vaccines, and Viruses Journals Mr. Marquette Pate: Partially joined this project. Animal Science major (non-thesis category), conducted his senior thesis in our laboratory involving this project, and continued his graduate study in our Food and Animal Science Master Program from Fall 2020. Ms. Alessandria Aikerson-Russell: Partially joined this project. Animal Science major (non-thesis category), started her graduate study in our Food and Animal Science Master Program from Spring 2020. Two Postdoctoral Researchers: Dr. Yuanying Gong: a postdoctoral researcher sponsored by another project, assisted with handling PRRSV reverse genetics and antiviral assays Dr. Damarius Fleming:a postdoctoral researcher sponsored by USDA Postdoctoral Fellowship, assisted with handling animal tests and RNA-Seq analyses. How have the results been disseminated to communities of interest?We have disseminated the project results to communities through the following (but not limited to) pathways: • Publish our results mostly in peer-reviewed and open-access journals including Frontier Immunology, Lancet, Cells, Biomecules, Genes, Viruses, J Gen Viology, Vaccine, Pathogens • Present our results in on-campus/cross-campus seminars and class teaching in particular for DVM and graduate students in Veterinary Biomedical Sciences and animal sciences • Broadcast our findings through both classical (animal workers' gatherings such as on Swine Days) and online media (Such as PI and CO-PI websites through the Institutional Homepages) • Through the activity of TSU agricultural extension system, other local news media and online broadcasting (YouTube,etc.) • Others: a) Delivering presentations/seminars at the University annual Research Day/Forum; and other on-campus and intercampus presentations; b) exchange and publication of students' research theses; c) incorporation and introduction in lectures/labs in animal health/disease/immunology courses; and d) the department and inter-department seminar presentation, topic lectures or lab research training What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Obj. 1: (1) We have annotated IFN gene families across 110 animal genomes, and have shown that IFN genes (esp. IFN-omega subtype), after originating in jawed fishes, had several significant evolutionary surges in vertebrate species of amphibians, bats and ungulates, particularly pigs and cattle. (2) Pigs and cattle have the largest but still expanding type I IFN family consisting of nearly 60 IFN-coding genes that encode seven IFN subtypes including multigene subtypes of IFN-α, -δ and -ω. Whereas subtypes such as IFN-α and -β have been widely studied in many species, the unconventional subtypes such as IFN-ω have barely been investigated. (3) Genome-wide, family-wide and subtype-specific expression analysis in porcine cells and tissues in response to PRRSV/SIV infections Obj. 2 We have cross-species defined the IFN evolution, and shown that unconventional IFN subtypes particularly the IFN-ω subtype have evolved several novel features including: 1) being a signature multi-gene subtype expanding primarily in mammals such as bats and ungulates, 2) emerging isoforms that have superior antiviral potency than typical IFN-α, 3) highly cross-species antiviral (but little anti-proliferative) activity exerted in cells of humans and other mammalian species, 4) demonstrating potential novel molecular and functional properties, 5) Examine uncanonical signaling of IFNs via regulation of epigenetic and RAAS cascades. Obj. 3: Several porcine IFN-omega peptides have been expressed and purified to commercialize as research agents and used for producing immunoreagents. One IFN-omega isoform is in developing stage as a superior antiviral, and an IFN cohort is in the optimizing stage for adjuvant effect during a vaccine validation. By support from a relevant NIFA grant, we have performed animal tests for the promising effect of the proposed vaccine regimen.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sang ER, Tian Y, Miller LC, Sang Y. (2020) Epigenetic evolution of ACE2 and IL-6 genes as non-canonical interferon-stimulated genes correlates to COVID-19 susceptibility in vertebrates. Genes (Basel). 2021 Jan 25;12(2):154.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Lopez L, Sang PC, Pate M, Tian Y, Sang Y. (2020) Dysregulated Interferon Response Underlying Severe COVID19. Viruses, 2020, 12(12), 1433.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Fleming D, Miller LC, Tian Y, Li Y, Ma W, Sang Y. (2020) Impact of Porcine Reproductive and Respiratory Syndrome Virus, Influenza B, and Their Coinfection on Antiviral Response in the Porcine Lung. Pathogens, 2020, 9, 934.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: McCoy K, Peterson A., Tian Y, Sang Y. (2020) Immunogenetic Association Underlying Severe COVID19. Vaccines, 2020, 8(4), 700.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sang ER, Tian Y, Gong Y, Miller LC, Sang Y. (2020) Integrate structural analysis, isoform diversity, and interferon-inductive propensity of ACE2 to refine SARS-COV2 susceptibility prediction in vertebrates. Heliyon. 2020 Sep;6(9):e04818.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Dowson H, Sang Y, Lunney J. Porcine cytokines, chemokines and growth factors: 2019 Update. Res. Vet. Sci. 2020 Aug;131:266-300.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wu Y, Ho W, Huang Y, Jin D-Y, Li S, Liu S-L, Liu X, Qiu J, Sang Y, Wang Q, Yuen K-Y, Zheng Z-M (2020) SARS-CoV-2 is an appropriate name for the new coronavirus. The Lancet, 2020 Mar 21;395(10228):949-950.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Xiao C, Li X, Liu S, Sang Y, Gao S-J, & Gao F (2020) HIV-1 did not contribute to the 2019-nCoV genome. Emerging Microbes & Infections, 2020; 9:1, 378-381, DOI: 10.1080/22221751.2020.1727299
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Tian Y, DeJesus FA, Khwatenge C, Li J, Robert J, Sang Y. (2021) Virus-Targeted Transcriptomic and Functional Analyses Implicate Ranaviral Interaction with Host Interferon Response in Frog Virus 3-infected Frog Tissues. Viruses 2021, 13(7), 132.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Tian Y, Khwatenge C, Li J, DeJesus FA, Robert J, Sang Y. (2021) Virus-Targeted Transcriptomic Analyses in Frog Virus 3-infected Frog Tissues Reveal Non-Coding Regulatory Elements in Intergenic Regions of Ranaviral Genome and Their Molecular Interaction with Host Immune Response. Front Immunol. 2021 Jun 17;12:705253.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sang Y. (2019) Immunometabolic Links Underlying the Infectobesity with Persistent Viral Infections. J. Immunol. Sci. Aug, 2019; 3(4): 8-13.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sang Y. Shields LE, Sang RE, Si H, Pigg A, and Blecha F. (2019). Transcriptomic Analysis in Obese Rats Induced by High-Fat Diets plus an Adenoviral Infection. Int J Obes (Lond). 2019 Nov;43(11):2134-2142. doi: 10.1038/s41366-019-0323-2.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Tian Y, Jennings J, Gong Y, Sang Y. Viral Infections and Interferons in the Development of Obesity. Biomolecules. 2019 Nov 12;9(11). pii: E726. doi: 10.3390/biom9110726.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jennings J, Sang Y. (2019) Porcine Interferon Complex and Co-Evolution with Increasing Viral Pressure after domestication. Viruses 2019, 11(6), 555; https://doi.org/10.3390/v11060555
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Tian Y, Jennings J, Gong Y, Sang Y. (2019) Xenopus Interferon Complex: Inscribing the Amphibiotic Adaption and Species-Specific Pathogenic Pressure in Vertebrate Evolution? Cells. 2019 Dec 26;9(1). pii: E67.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Shields EL, Jennings J, Liu Q, Lee JH, Ma W, Miller CL, Blecha F, Sang Y. (2019). Cross-species genome-wide analysis reveals molecular and functional diversity of the unconventional interferon-? subtype. Frontier Immunology 2019 Jun 25;10:1431.
  • Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Li J, Sang ER, Adeyemi O, Miller LC, Sang Y. Comparative Transcriptome Reveals Small RNA Composition and Differential MicroRNA Responses Underlying Interferon-Mediated Antiviral Regulation in Porcine Alveolar Macrophages. Epigenetics, in review
  • Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Sarlo Davila KM, Miller LC, Sang Y, Nelli RK, Gimenez-Lirola LG. Transcriptomic analysis of porcine respiratory epithelial cells cultured in an air-liquid interface reveals mechanisms driving the immune response to the betacoronavirus PHEV. mBio, in submission
  • Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Adeyemi OD, Tian Y, Grayfer L, Sang Y. Molecular Diversity and Functional Implication of Amphibian Interferon Complex: Remarking Immune Adaptation in Vertebrate Evolution. Dev. Comp. Immunol., in review
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Fleming DS, Miller LC, Li J, Geelen AV, Sang Y. (2022). Transcriptomic Analysis of Liver Indicates Novel Vaccine to Porcine Reproductive and Respiratory Virus Promotes Homeostasis in T-Cell and Inflammatory Immune Responses Compared to a Commercial Vaccine in Pigs. Front. Vet. Sci., 24 March 2022 | https://doi.org/10.3389/fvets.2022.791034.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Khwatenge CN, Pata M, Miller LC, Sang Y. (2021) Immunometabolic Dysregulation at the Intersection of Obesity and COVID-19. Front Immunology 2021 Oct 19;12:732913. doi: 10.3389/fimmu.2021.732913.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Sang Y, Miller LC, Nelli RK, Gimenez-Lirola LG (2021) Harness Organoid Models for Virological Studies in Animals: A Cross-Species Perspective. Front Microbiology, 2021 Sep 16;12:725074. | https://doi.org/10.3389/fmicb.2021.725074


Progress 07/01/20 to 06/30/21

Outputs
Target Audience:Group I: Industry and academic professionals including swine immunologists, virologists, nutritionists, consultants and other researcher workers, as well as producers, veterinarians and veterinary researchers. Efforts: 1) Conference presentation at scientific meetings such as the Annual Conference of The American Association of Immunologists (AAI), International PRRS Symposium, and the Conference of Research Workers in Animal Diseases (CRWAD), International Nidovirus Symposium (Note: Although our abstracts have been accepted, several conferences were cancelled due to COVID19 crisis); 2) TSU's day at the capital city of Tennessee.; 3) publications in book chapters, and peer-reviewed and open-assessed scientific journals includingFrontier Immunology, Genes, Heliyon, Pathogens,Research inVeterinary Science, Viruses,Vaccine, 4) Cross-campus professional seminars in the national and international academic institutes (mostly online) Group II: Graduate and undergraduate students. Efforts: 1) In addition to the first effort above, data/discoveries have also been presented specifically at the University annual Research Day/Forum; and 2) other in-campus and inter-campus student research forums; 3) exchange and publication of students' research theses; 4) incorporation and introduction in lectures/labs of animal health/disease/immunology courses; 5) the department and inter-department seminar presentation, topic lectures or lab research training Group III: Other types of public audience. Efforts: Through the activity of TSU agricultural extension system, other local news media and online broadcasting (YouTube, Laboratory and the PI's Institutional Website etc.) Changes/Problems:A non-cost extension was submitted and approved for efficient use of the remaining budget, training students and finalizing results/data for publication and dissemination. Otherwise, no major changes/problems are needed or have met so far except some unexpected delay due to COVID-19 shut-downs. What opportunities for training and professional development has the project provided?Three undergraduate research assistants: Ms. Kendall McCoy: Biological Sci Major, TLSAMP Scholarship Student. Joined the project in June, 2020, submitted an abstract to TLSAMP Conference and co-authored a relevant paper in the Vaccines Journal Ms. Autumn Peterson: Biological Sci Major, TLSAMP Scholarship Student. Joined the project in June, 2020, submitted an abstract to TLSAMP Conference and co-authored a relevant paper in the Vaccines Journal Ms. LeAnn Lopez: Animal Sci Major, Dean Scholarship Student. Joined the project in April, 2020, submitted an abstract to TLSAMP Conference, co-authored a relevant paper in the Vaccines Journal and co-authored a relevant paper in the Viruses Journal Four graduate students: Mr. Jordan Jennings: Joined this project from January to September 2020, and successfully graduated from Food and Animal Science Master Program with five papers published in Frontier Immunology, Cells, Biomolecules, Viruses; he was immediately hired after graduationas Research Scientist at Vanderbilt University Medical School Ms. Yun Tian: Partially joined this project during this entire reporting period, submitted an abstract to 2020 CORWAD Conference for student research award competition and co-authored four relevant papers in Hellion, Pathogens and Vaccines, and Viruses Journals Mr. Marquette Pate: Partially joined this project. Animal Science major (non-thesis category), conducted his senior thesis in our laboratory involving this project, and continued his graduate study in our Food and Animal Science Master Program from Fall 2020. Ms. Alessandria Aikerson-Russell: Partially joined this project. Animal Science major (non-thesis category), started her graduate study in our Food and Animal Science Master Program from Spring 2020. One Postdoctoral Researcher: Dr. Yuanying Gong: a postdoctoral researcher sponsored by another project, assisted with handling PRRSV reverse genetics and antiviral assays How have the results been disseminated to communities of interest?We have disseminated the project results to communities through the following (but not limited to) pathways: • Publish our results mostly in peer-reviewed and open-access journals including Frontier Immunology, Lancet, Cells, Biomecules, Genes, Viruses, J Gen Viology, Vaccine, Pathogens • Present our results in on-campus/cross-campus seminars and class teaching in particular for DVM and graduate students in Veterinary Biomedical Sciences and animal sciences • Broadcast our findings through both classical (animal workers' gatherings such as on Swine Days) and online media (Such as PI and CO-PI websites through the Institutional Homepages) • Through the activity of TSU agricultural extension system, other local news media and online broadcasting (YouTube,etc.) • Others: a) Delivering presentations/seminars at the University annual Research Day/Forum; and other on-campus and inter-campus presentations; b) exchange and publication of students' research theses; c) incorporation and introduction in lectures/labs in animal health/disease/immunology courses; and d) the department and inter-department seminar presentation, topic lectures or lab research training What do you plan to do during the next reporting period to accomplish the goals?We have almost completed Objectives 1 & 2, the effort in the next period for Objective 2 will be to complete and publish the results of molecular identification and phylogenic analyses of porcine IFN-omega molecules and do subtype comparison across livestock species. Additional work during the next reporting period will primarily focus on functional characterization of porcine IFN-omega per IFN signaling specificity. We have conducted a whole transcriptomic analysis for the virus-infected macrophages to profile all RNA species including both mRNA and regulatory small RNA, and the data is in processing/analysis for publication. For Objective 2. We will express more IFN peptides or use the approaches of genetic manipulation to examine antiviral activity or other biological function of selected IFN-omega peptides in our virus-cell models. For Objective 3, we will keep developing and coordinating for marketing the IFN peptides (or constructs or molecular assays) and validating a vaccine candidate, which is potentially applicable in animal science research and antiviral therapies in biomedicine. In addition, we are processing to file one to two-relevant Patents. This is the term of no-cost extension, we will focus on data processing and publishing, and conceive novel ideas for further investigation.

Impacts
What was accomplished under these goals? For Obj. 1: (1) We have annotated IFN gene families across 110 animal genomes, and have shown that IFN genes (esp. IFN-omega subtype), after originating in jawed fishes, had several significant evolutionary surges in vertebrate species of amphibians, bats and ungulates, particularly pigs and cattle. (2) Pigs and cattle have the largest but still expanding type I IFN family consisting of nearly 60 IFN-coding genes that encode seven IFN subtypes including multigene subtypes of IFN-α, -δ and -ω. Whereas subtypes such as IFN-α and -β have been widely studied in many species, the unconventional subtypes such as IFN-ω have barely been investigated. (3) Genome-wide, family-wide and subtype-specific expression analysis in porcine cells and tissues in response to PRRSV/SIV infections For Obj. 2 We have cross-species defined the IFN evolution, and shown that unconventional IFN subtypes particularly the IFN-ω subtype have evolved several novel features including: 1) being a signature multi-gene subtype expanding primarily in mammals such as bats and ungulates, 2) emerging isoforms that have superior antiviral potency than typical IFN-α, 3) highly cross-species antiviral (but little anti-proliferative) activity exerted in cells of humans and other mammalian species,4) demonstrating potential novel molecular and functional properties, 5) Examine uncanonical signaling of IFNs via regulation ofepigenetic and RAAS cascades. For Obj. 3: Several porcine IFN-omega peptides have been expressed and purified to commercialize as research agents and used for producing immunoreagents. One IFN-omega isoform is in developing stageas a superior antiviral, and an IFN cohort is in theoptimizing stagefor adjuvant effect during a vaccine validation. By support from a relevantNIFA grant,we have performed animal tests for the promising effect of the proposed vaccine regimen.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Harness Organoid Models for Validation of Animal Viruses: A Cross- Species Perspective. Cells (In review)
  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Tian Y, Khwatengea C, Li J, DeJesus FA, Robert J, Sang Y. (2021) Virus-targeted Transcriptomic Analyses in Frog Virus 3-infected Frog Tissues Reveal Non-Coding Regulatory Elements in Intergenic Regions of Ranaviral Genome and Their Molecular Interaction with Host Immune Response. Front Immunology (Accepted)
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Tian Y, DeJesus FA, Khwatengea C, Li J, Robert J, Sang Y. (2021) Virus-Targeted Transcriptomic Analyses Implicate Ranaviral Interaction with Host Interferon Response in Frog Virus 3-infected Frog Tissues. Viruses (In review)
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sang ER, Tian Y, Miller LC, Sang Y. (2020) Epigenetic evolution of ACE2 and IL-6 genes as non-canonical interferon stimulated genes correlates to COVID-19 susceptibility in vertebrates. Genes (Basel). 2021 Jan 25;12(2):154.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Lopez L, Sang PC, Pate M, Tian Y, Sang Y. (2020) Dysregulated Interferon Response Underlying Severe COVID19. Viruses, 2020, 12(12), 1433.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Fleming D, Miller LC, Tian Y, Li Y, Ma W, Sang Y. (2020) Impact of Porcine Reproductive and Respiratory Syndrome Virus, Influenza B, and Their Coinfection on Antiviral Response in the Porcine Lung. Pathogens, 2020, 9, 934.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: McCoy K, Peterson A., Tian Y, Sang Y. (2020) Immunogenetic Association Underlying Severe COVID19. Vaccines, 2020,8(4), 700.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sang ER, Tian Y, Gong Y, Miller LC, Sang Y. (2020) Integrate structural analysis, isoform diversity, and interferon inductive propensity of ACE2 to refine SARS-COV2 susceptibility prediction in vertebrates. Heliyon, 2020 Sep;6(9):e04818.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Dowson H, Sang Y, Lunney J. Porcine cytokines, chemokines and growth factors: 2019 Update. Res. Vet. Sci. 2020 Aug;131:266-300.


Progress 07/01/19 to 06/30/20

Outputs
Target Audience:Group I: Industry and academic professionals including swine immunologists, virologists, nutritionists, consultants and other researcher workers, as well as producers, veterinarians and veterinary researchers. Efforts: 1) Conference presentation at scientific meetings such as the Annual Conference of The American Association of Immunologists (AAI), International PRRS Symposium, and the Conference of Research Workers in Animal Diseases (CRWAD), International Nidovirus Symposium (Note: Although our abstracts have been accepted, several conferences were cancelled due to COVID19crisis); 2) TSU's day at the capital city of Tennessee.; 3) publications in book chapters, and peer-reviewed and open-assessed scientific journals including Emerging Microbes & Infections, Lancet, Biomecules; Cells, Frontier Immunology, Viruses; 4) Cross-campus professional seminars in the national and international academic institutes. Group II: Graduate and undergraduate students. Efforts: 1) In addition to the first effort above, data/discoveries have also been presented specifically at the University annual Research Day/Forum; and 2) other in-campus and inter-campus student research forums; 3) exchange and publication of students' research theses; 4) incorporation and introduction in lectures/labs of animal health/disease/immunology courses; 5) the department and inter-department seminar presentation, topic lectures or lab research training Group III: other interested audiences. Efforts: Through the activity of TSU agricultural extension system, other local news media and online broadcasting (YouTube, etc.) Changes/Problems:A non-cost extension was submitted and approved for efficient use of the remaining budget, training students and finalizing results/data for publication and dissemination Otherwise, no major changes/problems are needed or have met so far. What opportunities for training and professional development has the project provided?Two undergraduate research assistants: Jory Teague has been working in this program for the semester of Spring & Summer, 2019, graduated in this May, 2019. He is now accepted as a graduate student sponsored by TSU Dean Graduate Scholarship . Simen Asefaw is a junior student at our PreVet major, and start working this project part-timely from Feb, 2020. Three graduate students: (3) Jordan Jennings (TSU), a master student is now full-timely working in this project since Spring,2019, and has accomplished his master thesis as a part of this project, and currently are looking for a job or keep going with Ph.D studying in biomedical field. (4) Yun Tian (TSU), a Ph.D student helps viral/cell handling with this project (5) Liping Wang (KSU) has started her PhD program in the fall 2017, and contributes this project from Spring 2019 (6) Nirmalendu Deb (KSU) Nath has started his master program in the Spring 2018, and partly working on this project from Fall 2019 Two Postdoctoral Researchers: (1) Dr. Jinhwa Li, a postdoctoral researcher, has been partially worked on this project to test antiviral activity of some antiviral genes. She has two joint publications/manuscripts relevant to this project (2) Dr. Yuanying Gong, a postdoctoral researcher, helps for handling PRRSV reverse genetics and antiviral assays How have the results been disseminated to communities of interest?We have disseminated the project results to communities through the following pathways: (1) Publish our results mostly in peer-reviewed and open-access journals including Frontier Immunology, Lancet, Cells, Biomecules, Viruses, J Gen Viology (2) Present our results in on-campus seminars and class teaching in particular for DVM and graduate students in Veterinary Biomedical Sciences and animal sciences (3) Broadcast our discoveries through both classical (animal workers' gathers such as in Swine Days) and online medias (such as http://www.k-state.edu/media/newsreleases/jul16/amphibians71516.html http://veterinarynews.dvm360.com/kansas-state-researchers-investigate-threat-influenza-amphibians https://www.ars.usda.go/research/publications/publication/?seqNo115=327085 https://scholar.google.com/citations?hl=en&user=mV-iDZMAAAAJ&view_op=list_works&sortby=pubdatev (4) Through the activity of TSU agricultural extension system, other local news media and online broadcasting (YouTube, etc.) (5) Others: a) Delivering presentations/seminars at the University annual Research Day/Forum; and other in-campus and inter-campus presentations; b) exchange and publication of students' research theses; c) incorporation and introduction in lectures/labs of animal health/disease/immunology courses; and d) the department and inter-department seminar presentation, topic lectures or lab research training What do you plan to do during the next reporting period to accomplish the goals?We have pretty much done with the Objective 1 & 2, the effort in the next period for Objective 2 is to complete and publish the results of molecular identification and phylogenic analyses of porcine IFN-omega molecules and do subtype comparison across livestock species. The further work of the next reporting period will primarily focus on functional characterization of porcine IFN-omega per IFN signaling specificity. For Objective 2. We will express more IFN peptides or use the approaches of genetic manipulation to examine antiviral activity or other biological function of selected IFN-omega peptides in our virus-cell models. For Objective 3, we will keep developing and coordinate for marketing the IFN peptides (or constructs or molecular assays) and validating a vaccine candidate, which potentially applicable animal science research and antiviral therapies in biomedicine. In addition, we are processing to file two-relevant Patents

Impacts
What was accomplished under these goals? For Obj. 1: (1) We have annotated IFN gene families across 110 animal genomes, and showed that IFN genes (esp. IFN-omega subtype), after originating in jawed fishes, had several significant evolutionary surges in vertebrate species of amphibians, bats and ungulates, particularly pigs and cattle. (2) Pigs and cattle have the largest but still expanding type I IFN family consisting of nearly 60 IFN-coding genes that encode seven IFN subtypes including multigene subtypes of IFN-α, -δ and -ω. Whereas subtypes such as IFN-α and -β have been widely studied in many species, the unconventional subtypes such as IFN-ω have barely been investigated. (3) Genome-wide, family-wide and subetype-specific expression analysis in porcine cells and tissues in response to PRRSV/SIV infections For Obj. 2 We have cross-species defined the IFN evolution, and shown that unconventional IFN subtypes particularly the IFN-ω subtype have evolved several novel features including: 1) being a signature multi-gene subtype expanding primarily in mammals such as bats and ungulates, 2) emerging isoforms that have superior antiviral potency than typical IFN-α, 3) highly cross-species antiviral (but little anti-proliferative) activity exerted in cells of humans and other mammalian species, and 4) demonstrating potential novel molecular and functional properties. For Obj. 3: Several porcine IFN-omega peptides have been expressed and purified to commerilize as research agents and used for producing immunoreagents. One IFN-omega isoform is in developing as a superior antiviral, and an IFN cohort is optimizing for adjuvant effect during a vaccine validation

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 1. Dowson H, Sang Y, Lunney J. Porcine cytokines, chemokines and growth factors: 2019 Update. Res. Vet. Sci. May 2020, 131:266-300. DOI: 10.1016/j.rvsc.2020.04.022 PMID: 32442727.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 2. Wu Y, Ho W, Huang Y, Jin D-Y, Li S, Liu S-L, Liu X, Qiu J, Sang 2. Wu Y, Wang Q, Yuen K-Y, Zheng Z-M (2020) SARS-CoV-2 is an appropriate name for the new coronavirus. The Lancet, Published Online March 5, 2020 https://doi.org/10.1016/S0140-6736(20)30557-2
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 3. Xiao C, Li X, Liu S, Sang Y, Gao S-J, & Gao F (2020) HIV-1 did not contribute to the 2019-nCoV genome. Emerging Microbes & Infections, 2020; 9:1, 378-381, DOI: 10.1080/22221751.2020.1727299
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 4. Sang Y. (2019) Immunometabolic Links Underlying the Infectobesity with Persistent Viral Infections. J. Immunol. Sci. Aug, 2019; 3(4): 8-13.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 5. Sang Y. Shields LE, Sang RE, Si H, Pigg A, and Blecha F. (2019). Transcriptomic Analysis in Obese Rats Induced by High-Fat Diets plus an Adenoviral Infection. Int J Obes (Lond). 2019 Nov;43(11):2134-2142. doi: 10.1038/s41366-019-0323-2.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 6.Tian Y, Jennings J, Gong Y, Sang Y. Viral Infections and Interferons in the Development of Obesity. Biomolecules. 2019 Nov 12;9(11). pii: E726. doi: 10.3390/biom9110726.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 7. Tian Y, Jennings J, Gong Y, Sang Y. (2019) Xenopus Interferon Complex: Inscribing the Amphibiotic Adaption and Species-Specific Pathogenic Pressure in Vertebrate Evolution? Cells. 2019 Dec 26;9(1). pii: E67
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 8. Jennings J, Sang Y. (2019) Porcine Interferon Complex and Co-Evolution with Increasing Viral Pressure after domestication. Viruses 2019, 11(6), 555; https://doi.org/10.3390/v11060555
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 9. Shields EL, Jennings J, Liu Q, Lee JH, Ma W, Miller CL, Blecha F, Sang Y. (2019). Cross-species genome-wide analysis reveals molecular and functional diversity of the unconventional interferon-? subtype. Frontier Immunology 2019 Jun 25;10:1431
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 10. Lee J, Wang L, Palinski R, Walsh T, He D, Li Y, Wu R, Lang Y, Sunwoo SY, Richt JA, Ma W. Comparison of pathogenicity and transmissibility of influenza B and D viruses in pigs. Viruses. 2019. 11(10). PMID: 31569752
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Damarius S. Fleming, Laura C. Miller, Yun Tian, Yonghai Li, Wenjun Ma, Yongming Sang. Impact of PRRSV, Influenza B, and Their Coinfection on Antiviral Response in the Porcine Lung. NIDO2020 - the XVth INTERNATIONAL NIDOVIRUS SYMPOSIUM Egmond aan Zee, The Netherlands, May 10-14, 2020


Progress 07/01/18 to 06/30/19

Outputs
Target Audience:Group I: Industry and academic professionals including swine immunologists, virologists, nutritionists, consultants and other researcher workers, as well as producers, veterinarians and veterinary researchers. Efforts: 1) Conference presentation at scientific meetings such as the Annual Conference of The American Association of Immunologists (AAI), International PRRS Symposium, and the Conference of Research Workers in Animal Diseases (CRWAD); 2) TSU's day at the capital city of Tennessee.; 3) publications in book chapters, and peer-reviewed and open-assessed scientific journals including Plos One, J Gen Virol, J Virol, Frontier Immunology, Viruses, J. Immunol.; 4) Cross-campus professional seminars innational and international academic institutes. Group II: Graduate and undergraduate students. Efforts: 1) In addition to the first effort above, the data will also be presented specifically at the University annual Research Day/Forum; and 2) other in-campus and inter-campus student research forums; 3) exchange and publication of students' research theses; 4) incorporation and introduction in lectures/labs of animal health/disease/immunology courses; 5) the department and inter-department seminar presentation, topic lectures or lab research training Group III: other interested audiences. Efforts: Through the activity of TSU agricultural extension system, other local news media and online broadcasting (YouTube, etc.) Changes/Problems:No major changes/problems are needed or have met so far. What opportunities for training and professional development has the project provided?Two undergraduate research assistants: (1) Pat Marquette had worked in our laboratory for 0.5 year, graduated and left our laboratory in December, 2018. He is currently a graduate student in UTK poultry science (2)Jory Teaguehas been working in this program forthe semester of Spring, 2019, graduated in this May. He is now accepted as a graduate student sponsored by TSU Dean Graduate Scholarship . Threegraduate students: (1) Lauren E. Shields, a master student partially worked on this project to test IFN-PRRSV interaction, she has graduated in May, 2019 (2) Jordan Jennings, a master student is now full-timely working in this project since Spring, 2019 (3) Yun Tian, a Ph.D student helps viral/cell handling with this project Two Postdoctoral Researchers: (1) Dr. Jinhwa Li, apostdoctoral researcher, has been partially worked on this project to test antiviral activity of some antiviral genes. She has two joint publications/manuscripts relevant to this project (2) Dr. Yuanying Gong, a postdoctoral researcher, helps for handling PRRSV reverse genetics and antiviral assays How have the results been disseminated to communities of interest?We have disseminated the project results to communities through the following pathways: (1) Publish our results mostly in peer-reviewed and open-access journals including Frontier Immunology,Viruses, J Gen Viology (2) Present our results in on-campus seminars and class teaching in particular for DVM and graduate students in Veterinary Biomedical Sicences and animal sciences (3) Broadcast our discoveries through both classical (animal workers' gathers such as in Swine Days) and online medias (such as http://www.k-state.edu/media/newsreleases/jul16/amphibians71516.html http://veterinarynews.dvm360.com/kansas-state-researchers-investigate-threat-influenza-amphibians https://www.ars.usda.go/research/publications/publication/?seqNo115=327085 https://scholar.google.com/citations?hl=en&user=mV-iDZMAAAAJ&view_op=list_works&sortby=pubdatev (4) Through the activity of TSU agricultural extension system, other local news media and online broadcasting (YouTube, etc.) (5) Others: a) Delieving presentations/seminarsat the University annual Research Day/Forum; andother in-campus and inter-campus presentations; b) exchange and publication of students' research theses; c) incorporation and introduction in lectures/labs of animal health/disease/immunology courses; and d) the department and inter-department seminar presentation, topic lectures or lab research training What do you plan to do during the next reporting period to accomplish the goals?We have pretty much done with the Objective 1 & 2, the effort in the next period for Objective2 is to complete and publish the results of molecular identification and phylogenic analyses of porcine IFN-omega molecules and do subtype comparisonacross livestock species. The further work of the next reporting period will primarily focus on functional characterization ofporcine IFN-omega per IFN singaling specificity. For Objective 2. We will express more IFN peptides or use the approaches of genetic manipulation to examine antiviral activity or other biological function of selected IFN-omega peptides in our virus-cell models. For Objective 3, we will keep developing and coordinate for marketing the IFN peptides (or constructs or molecular assays) which potentially applicable animal science research and antiviral therapies in biomedicine. In addition, we are processing tofiletwo-relevantPatents

Impacts
What was accomplished under these goals? For Obj. 1: (1) We have annotated IFN gene families across 110 animal genomes, and showed that IFNgenes (esp. IFN-omega subtype), after originating in jawed fishes, had several significant evolutionary surges in vertebrate species of amphibians, bats and ungulates, particularly pigs and cattle. (2) Pigs and cattlehave the largest but still expanding type I IFN family consisting of nearly 60 IFN-coding genes that encode seven IFN subtypes including multigene subtypes of IFN-α, -δ and -ω. Whereas subtypes such as IFN-α and -β have been widely studied in many species, the unconventional subtypes such as IFN-ω have barely been investigated. (3) Family-wide and subetype-specific expression analysis in porcine cells andtissues in response to PRRSV/SIV infections For Obj. 2 We have cross-species defined the IFN evolution, and shown that unconventional IFN subtypes particularly the IFN-ω subtype have evolved several novel features including: 1) being a signature multi-gene subtype expanding primarily in mammals such as bats and ungulates, 2) emerging isoforms that have superior antiviral potency than typical IFN-α, 3) highly cross-species antiviral (but little anti-proliferative) activity exerted in cells of humans and other mammalian species, and 4) demonstrating potential novel molecular and functional properties. For Obj. 3: Several porcine IFN-omega peptides have been expressed and purified to commerilize as research agents and used for producing immunoreagents. One IFN-omega isoform is in developing as an superior antiviral

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Comparison of the transcriptome response within the porcine tracheobronchial lymph node following viral infection. World Congress of Genetics Applied in Livestock Production 2018, Conference proceedings, 5/10/2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Small non-coding RNA expression status in animals faced with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). World Congress of Genetics Applied in Livestock Production 2018, Conference proceedings, 5/10/2018
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sang Y. Shields LE, Sang RE, Si H, Pigg A, and Blecha F. (2019). Transcriptomic Analysis in Obese Rats Induced by High-Fat Diets plus an Adenoviral Infection. Int J Obes (Lond). 2019 Jan 22. doi: 10.1038/s41366-019-0323-2. [Epub ahead of print]
  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Shields EL, Jennings J, Liu Q, Lee JH, Ma W, Miller CL, Blecha F, Sang Y. (2019). Molecular and functional novelty of porcine interferon-omega subtypes. Frontier Immunology (in press, Corresponding author).
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Jennings J, Sang Y. (2019) Porcine Interferon Complex and Co-Evolution with Increasing Viral Pressure. Viruses (Accepted, review per editor solicitation)
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Sang Y. (2019) Viral Infections and Obesity: Interferons Come into Play. Cells (In preparation, review per editor solicitation)