THE ROLE OF THE INNATE IMMUNE RESPONSE IN RESISTANCE TO VIRAL INFECTION AND DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0219901
• Project Start Date: Oct 1, 2009
• Project End Date: Sep 30, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Poultry Science

Non Technical Summary
Non-Technical Summary Understanding how cells of the infected animal first detect the presence of viral infection, how that initial detection may affect the type of immune response, and how the immune response affects the overall clinical outcome, is essential to our ability to produce novel vaccine and control strategies for various viral infections. We expect the results from this Animal Health Project will lead to the identification of a few key genes involved in this immediate-early detection of infection and which determine the course of the response. The identification of these genes will allow us to develop methods to augment this initial response and enhance the overall host response and/or develop breeding strategies to select for animals with enhance resistance to infection.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3299	1101	40%
311	3299	1090	40%
311	3299	1080	20%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1090 - Immunology; 1101 - Virology; 1080 - Genetics;

Keywords

antiviral

innate immunity

genetic resistance

poultry

astrovirus

coronavirus

influenza virus

Goals / Objectives
Goals/Objectives/Expected Outputs 1) Identify innate immune factors associated with immunity and resistance to enteric infections. Following viral infection in vivo the cells of the infected tissue recognize the presence of virus and upregulate specific early innate immune mediators such as type I interferon and other chemokines and cytokines. The expression of these different factors stimulates cells of the innate immune system (such as macrophages and heterophils), resident in the tissue as well as those located distally, to participate in the response. The specific host factors upregulated immediately following detection of the pathogen play a major role in determining the type of immune response that occurs and may also affect the severity of the clinical disease. Understanding which host factors are associated with this immediate-early response and their role in the influencing the immune response which ultimately leads to clearance of the pathogen is essential to our ability to develop more effective therapies to promote animal health. 2) Identify genes and gene networks associated with intracellular recognition of viral infection and initiation of the antiviral host response. Within normal host cells there are specific proteins that function as sentries for the presence of pathogens. The more rapidly these factors recognize the presence of a viral infection, the quicker the host is capable of initiating the response to infection. Our understanding of these host factors is limited, especially in agricultural species. Recent studies in our laboratory suggest there are differences in the function of these various pathogen detection proteins among different genetic lines of poultry. Understanding the genetic basis for the differences in the response to viral infection is essential to our ability to understand how these different factors function, their relative importance in the response to different pathogens, and out ability to develop breeding strategies to enhance the resistance of animals to infection.

Project Methods
Methods Objective 1. Identify innate immune factors associated with immunity and resistance to enteric infections. Naive turkey poults will be infected with turkey astrovirus type-2 (TAstV-2) or other enteric viruses and the change in expression of genes associated with both innate and adaptive immunity assayed by real time RT-PCR. Specifically samples will be analyzed for expression of IL-1b, IL-2, IL-6, IL-10, IFNa, IFNg, iNOS, Mx, and 2'-5' OAS. Changes in expression will be determined using the ΔΔCT method and 18S ribosomal RNA as an internal standard. As additional relevant turkey genes are identified and sequenced we will expand our analysis. The biological activity and function of innate immune cells (such as macrophages and heterophils) will be assessed ex vivo to correlate the expression of various immune response inducing factors with activation of the innate immune response. Tissue sections will be collected from the site of infection and assayed by immunohistochemistry for the presence of innate immune cells as well as factors produced by innate immune cells. Innate immune cells will also be isolated from various compartments (peripheral blood, spleen, abdomen, etc) and cultured ex vivo and examined for differences in functional properties (adherence, production of cytotoxic mediators, phagocytosis, and gene expression). Objective 2. Identify genes and gene networks associated with intracellular recognition of viral infection and initiation of the antiviral host response. Our laboratory has previously developed a panel of approximately 50 primary chicken embryo fibroblast cell lines with distinct genotypes. These cell lines were derived from commercial layer-type chickens. We have worked to characterize the phenotypic differences in the response to viral infection among these lines with multiple different viruses. These studies have identified differences in the relative resistance and susceptibility to viral infection. Genes and gene networks associated these differences in resistance will be assessed using targeted oligo-nucleotide arrays to assay for fold differences in gene expression. The targeted arrays will allow for the simultaneous analysis of approximately 500 genes. Changes in gene expression will be compared within cell lines (infected vs control), across uninfected cell lines, and across infected cell lines. Analyses of these data will identify genes and gene families associated with resistance.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience:During the last reporting period we have targeted scientist as our audience via national level meeting and workshops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results have been disseminated to the broader scientific community through journal publications and presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue these research projects as vehicles for continued insights to understand innate immunity in chickens and to achieve the goals of this project.

Impacts
What was accomplished under these goals? During the previous reporting period, we have worked and made considerable progress on both the goal/objectives for this project. We have continued to work towrds understanding now the intestine recognizes and responds to bacteria and viruses. We have generated considerable data characterizing the microbiome of the chicken intestine and how augmenting the bacterial ecology affects the host. With respect to objective 2 we have characterized the genetic diversity of a specific innate immune gene (Mx) with in a library of elite layer-type chickens.

Publications

• Type: Book Chapters Status: Published Year Published: 2013 Citation: S. Guix, N. Krishna, and M. D. Koci. Astrovirus Immunity. Astrovirus Research: Essential Ideas, Everyday Impacts, Future Directions. Ed S. Schultz-Cherry and E. Mendez. Springer Science. New York. 2013.
• Type: Book Chapters Status: Published Year Published: 2013 Citation: M. Pantin-Jackwood, D. Todd, and M. D. Koci. Avian Astroviruses. Astrovirus Research: Essential Ideas, Everyday Impacts, Future Directions. Ed S. Schultz-Cherry and E. Mendez. Springer Science. New York. 2013.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: J.E. Fulton, J. Arango, M. Koci, and C.M. Ashwell. Variation in the MX gene in commercial egg layer elite lines. September 2013. 8th European Symposium on Poultry Genetics. Venice, Italy.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: W. J. Croom, C. W. Nash, M. Koci, B. W. McBride, B. Clemmons and J. T. Brake. Increasing growth in broilers by serotonin modulation: The effects of selective serotonin reuptake inhibitors. September 2013. Innovate 2013. Braselton, GA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: A. L. Ballou, R. Qiu, R. A. Ali, W. J. Croom, and M. D. Koci. Direct fed microbial supplementation affects host immune function and energy consumption. April 2013. Experimental Biology 2013. Boston, MA.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Animal scientists and allied industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have continued to develop methods and reagents to specifically study the innate immune response in poultry species to enteric infections and identify means of preventing diseases.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Peptides. 2013 Jul 26;48C:96-105. doi: 10.1016/j.peptides.2013.07.014. [Epub ahead of print] A mastoparan-derived peptide has broad-spectrum antiviral activity against enveloped viruses. Sample CJ, Hudak KE, Barefoot BE, Koci MD, Wanyonyi MS, Abraham S, Staats HF, Ramsburg EA.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: We have worked for the past several years to understand the host response the type-2 turkey astrovirus (TAstV-2). We have previously demonstrated TAstV-2 induces an acute diarrhea in turkey poults, that is associated with changes in the apical expression of the sodium/hydrogen exchanger-3 (NHE3). More recently in collaboration with the Schultz-Cherry laboratory at St. Jude Children's Research Hospital, we have examined the specific elements of the TAstV-2 virus that induce diarrhea. In these studies we demonstrated that oral inoculation of turkey poults with recombinantly expressed TAstV-2 capsid protein is sufficient to induce diarrhea, changes in intestinal barrier function, and changes in NHE3 activity. PARTICIPANTS: Stacey Schultz-Cherry St. Jude Children's Research Hospital TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
These findings provide a better understanding of how astroviruses induce disease in poultry.

Publications

• R. R. Meyerhoff, R. A. Ali, K. Liu, G. Q. Huang, M. D. Koci. Comprehensive analysis of commercially available mouse antichicken monoclonal antibodies for cross-reactivity with peripheral blood leukocytes from commercial turkeys. Poultry Science. 91:383-92, 2012.
• Regulation of the immune response through energy. What role does the microbiota play 2012 Eastern Nutrition Conference. Kitchener, Ontario, Canada. May 2012.
• S. Guix, N. Krishna, and M. D. Koci. Astrovirus Immunity. Astroviruses. Ed S. Schultz-Cherry and E. Mendez. Springer Science. (In Press) 2012.
• R. Qiu, J. Croom, R. A Ali, A. L. Ballou, C. Smith, C. M. Ashwell, H. M. Hassan, C.-C. Chiang, and M. D. Koci. Direct fed microbial supplementation repartitions host energy to the immune system. Journal of Animal Science. .90:1-13, 2012.
• R. R. Meyerhoff, P. K. Nighot, R. A. Ali, A. T. Blikslager, and M. D. Koci. Characterization of turkey inducible nitric oxide synthase and identification of its expression in the intestinal epithelium following astrovirus infection. Comparative Immunology, Microbiology, and Infectious Diseases. 35:63-9, 2012.
• M. Pantin-Jackwood, D. Todd, and M. D. Koci. Avian Astroviruses. Astroviruses. Ed S. Schultz-Cherry and E. Mendez. Springer Science. (In Press) 2012.
• Abstracts P. Nighot, R. Ali, L. A. Moser, P. Freiden, S. Schultz-Cherry, R. DuBois, A. Blikslager, and M. D. Koci. Turkey astrovirus capsid protein induces electrophysiological changes in the intestinal epithelium. August 2012. The 2012 American Association of Avian Pathologist/American Veterinary Medical Association Meeting. San Diego, CA.
• A. G. Seal, T. Helmy, R. Ali, E. Akers, M. Mendoza, E. Stafford, H. Hassan, and M. D. Koci. Development of a Salmonella specific ELISA to test the efficacy of a novel Salmonella vaccine. August 2012. 11th Annual NC State University Summer Undergraduate Research Symposium. Raleigh, NC.
• Y. T. Mak, M. Mendoza, T. Helmy, R. Ali, M. D. Koci, and H. Hassan. Efficacy of Salmonella detection in poultry: Evaluating different sampling methods to minimize number of animal use. August 2012. 11th Annual NC State University Summer Undergraduate Research Symposium. Raleigh, NC.
• J. Croom, C. Nash, M. D. Koci, J. Wock, A. Ballou, D. Carver, and J. Brake. Fluoxetine increases growth and feed efficiency in broilers. July 2012. Annual Meeting of the Poultry Science Association. Athens, GA.
• Direct fed microbial supplementation in poultry results in changes in both mucosal and systemic immune activity. 2012 Eastern Nutrition Conference. Kitchener, Ontario, Canada. May 2012.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: 1) Identify innate immune factors associated with immunity and resistance to enteric infections. Following viral infection in vivo the cells of the infected tissue recognize the presence of virus and upregulate specific early innate immune mediators such as type I interferon and other chemokines and cytokines. The expression of these different factors stimulates cells of the innate immune system (such as macrophages and heterophils), resident in the tissue as well as those located distally, to participate in the response. The specific host factors upregulated immediately following detection of the pathogen play a major role in determining the type of immune response that occurs and may also affect the severity of the clinical disease. Understanding which host factors are associated with this immediate-early response and their role in the influencing the immune response which ultimately leads to clearance of the pathogen is essential to our ability to develop more effective therapies to promote animal health. 2) Identify genes and gene networks associated with intracellular recognition of viral infection and initiation of the antiviral host response. Within normal host cells there are specific proteins that function as sentries for the presence of pathogens. The more rapidly these factors recognize the presence of a viral infection, the quicker the host is capable of initiating the response to infection. Our understanding of these host factors is limited, especially in agricultural species. Recent studies in our laboratory suggest there are differences in the function of these various pathogen detection proteins among different genetic lines of poultry. Understanding the genetic basis for the differences in the response to viral infection is essential to our ability to understand how these different factors function, their relative importance in the response to different pathogens, and out ability to develop breeding strategies to enhance the resistance of animals to infection. PARTICIPANTS: Collaborators Jim Croom Chris Ashwell Hosni Hassan TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
1) Findings have helped identify host factors which play a role in the innate response to enteric infections. These findings will result in the development of novel therapies which promote the expression of these host factors. 2) Findings have helped identify host cellular signaling pathways that regulate the innate immune response in the intestine and suggest mechanisms by wich changes in the host microflora may influence systemic immune responses.

Publications

• R. Meyerhoff, R. A. Ali, K. Liu, G-Q. Huang, and M. D. Koci. Comprehensive Analysis of Commercially Available Mouse Anti-Chicken Monoclonal Antibodies for Cross-Reactivity with Peripheral Blood Leukocytes from Commercial Turkeys. Poultry Science. 2012 91(2):383-392
• R. Qiu, J. Croom, R. A. Ali, C. Ashwell, H. M. Hassan, C. C. Chiang, and M. D. Koci. Direct Fed Microbial Supplementation Repartitions Host Energy to the Immune System. Journal of Animal Science. In Press. 2012.
• R. R. Meyerhoff, P. K. Nighot, R. A. Ali, A. T. Blikslager, and M. D. Koci. Characterization of Turkey Inducible Nitric Oxide Synthase and Identification of its Expression in the Intestinal Epithelium Following Astrovirus Infection. Comparative Immunology, Microbiology, and Infectious Diseases. 2012 35(1):63-9.
• N. M. Leandro, R. Ali, M. Koci, V. Moraes, R. Malheiros, M. J. Wineland, and E. O. Oviedo-Rondon. Effects of Broiler Breeder Genetics, Diet Type, and Feeding Program on Maternal Antibody Transfer and Development of Lymphoid Tissues. Journal of Applied Poultry Research. 20:474-484. 2011.
• N. M. Leandro, R. Ali, M. Koci, V. Moraes, P. E. Eusebio-Balcazar, J. Jornigan, R. D. Malheiros, M. J. Wineland, J. Brake, and E. O. Oviedo-Rondon. Maternal Antibody Transfer to Broiler Progeny Varies Among Strains and Is Affected by Grain Source and Cage-Density. Poultry Science. 90:2730-2739. 2011.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Development of turkey specific anti-iNOS antibodies as well as the establishment of methods using these reagents to detect the expression of iNOS in fixed turkey tissues. Development of turkey specific anti-CXCL8 antibodies and expression systems to produce turkey CXCL8 recombinantly in bacteria cells. PARTICIPANTS: Chris Ashwell, NC State Anthony Blikslager, NC State Jim Croom, NC State Jesse Grimes, NC State TARGET AUDIENCES: Scientific community North Carolina Poultry Industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The reagents and methods developed above will be critical to our ability to assay how the host responds to enteric viral challenge and how these changes are involved in the development of clinical diarrhea.

Publications

• P. K. Nighot, A. Moeser, R. A. Ali, A. T. Blikslager, and M. D. Koci. Astrovirus infection induces sodium malabsorption and redistributes sodium hydrogen exchanged expression. Virology. 401:146-54, 2010.
• J. L. Grimes, M. D. Koci, C. R. Stark, D. P. Smith, P. K. Nighot, and T. Middleton. Biological Effect of Naturally Occurring Mycotoxins Fed to Poults Reared to 21 Days of Age. International Journal of Poultry Science. 9: 871-874, 2010.