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United States Department of Agriculture

Agricultural Research Service

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2009 Annual Report

1a. Objectives (from AD-416)
The long-term goal of this project is to develop an improved understanding of genetic resistance and vaccinal responses to Marek’s disease (MD) in order to increase productivity and safety of poultry products. Over the next 5 years, we will focus on the following specific objectives: Objective 1: Curate and enhance the chicken genetic map and its integration with the genome sequence. Objective 2: Identify and characterize chicken genes and pathways that confer resistance to MD. Sub-objective 2A: Validate, fine-map, and identify positional candidate genes for quantitative trait loci (QTL) that confer resistance to MD. Sub-objective 2B: Evaluate non-major histocompatibility complex (MHC) host genomic effects on MD vaccine efficacy. Objective 3: Functional characterization of the chicken MHC in response to tumor-virus infection or vaccination. Sub-objective 3A: Determine the relationship between in vivo passage of Marek’s disease virus (MDV) and the emergence of MDV strains with increased virulence. Sub-objective 3B: Determine the relationship between chicken MHC genetics and virus evolution. Sub-objective 3C: Determine the molecular basis for differential levels of cell surface MHC class I glycoprotein expression.

1b. Approach (from AD-416)
We define three interrelated approaches to help achieve our goals. First, we continue to enhance and curate the East Lansing (EL) chicken genetic map, which provides the foundation for molecular genetic studies and the chicken genome assembly. Second, we use an integrative genomics approach to identify QTL and candidate genes that confer genetic resistance or vaccinal immunity to MD. Our efforts are greatly enhanced by the availability of characterized inbred and recombinant congenic strains (RCS), genetic markers from our map, comprehensive DNA microarrays, as well as infectious MDV-BAC clones that we can manipulate to query and characterize specific virus-host protein interactions. And third, we evaluate an in vivo model for MDV virulence evolution and if successful, ask the question whether increased virulence is restricted to specific major histocompatibility (MHC) haplotypes.

3. Progress Report
Substantial progress was made on all objectives. A high density DNA chip was developed that can score 56+K genetic markers simultaneously. This tool has been applied to evaluate DNA-based selection methods in commercial poultry, and to characterize genetic resistance to Marek’s disease (MD), a T cell lymphomas caused by the Marek’s disease virus (MDV) in commercial and experimental chickens. In addition, ultra high throughput sequencing has been incorporated to identify specific genes that response to MDV challenge. To provide more insights on MD control, we have developed an experimental method that mimics evolution of increased virulence of MDV strains. Specific genes and proteins have been investigated for their role in MDV pathogenesis and viral evasion of the immune system. The information and materials produced directly related to NP101 Component 1 as we have: (1) developed genome-enabling tools and reagents for the chicken, e.g., high-density genetic maps and 60K SNP genotyping chips, (2) identified a number of functional genes and their interactions both within the chicken and to MDV, especially as it relates to MD vaccines and virus evolution, and (3) have initiated efforts to critically evaluate genome-wide marker-assisted selection (GWMAS) in poultry.

4. Accomplishments
1. High density chicken DNA chip developed to enhance poultry breeding and fundamental knowledge. Poultry is the third largest agricultural commodity and primary meat consumed in the U.S. To meet the growing demands of consumers, the poultry industry will need to continue to improve methods of selection in breeding programs for production traits. The field of genomics offers great promise in providing fundamental information that can be readily translated into commercial applications. With the sequence (blueprint) of the chicken genome, the scientific challenge has shifted towards identifying genetic variation among individuals and how this contributes to differences in traits such as production. By targeted sequencing of commercial chickens, we designed a chip that can simultaneously score 56,702 genetic markers equally-spaced through the entire chicken genome. This powerful tool is being applied to identification of genes of agronomic importance, evaluation of the power of marker-based selection methods in commercial poultry, determination and preservation of genetic diversity.

2. Comprehensive screen for resistance genes to Marek’s disease in chicken. Marek’s disease (MD), a T cell lymphoma caused by the Marek’s disease virus (MDV), is the most severe chronic disease problem for the poultry industry due to unpredictable and spontaneous outbreaks in vaccinated flocks. To provide alternative and additional control measures, a comprehensive screen was conducted to identify chicken genes that respond to MDV infection and when found, determine if alleles within the genes respond differently. As a result, 3773 genes were identified that exhibited differential allele expression. If confirmed, this information can be used to directly select for chickens with enhanced resistance to MD, as well as provide a model for identifying genes for other economically-important traits.

3. Genome sequencing of chicken lines for Marek’s disease resistance in chicken. Genetic resistance to Marek’s disease, a costly problem to the poultry industry caused by a pathogenic virus, is desired as a way to augment vaccinal control. Unique inbred chicken lines that are resistant or susceptible to MD were sequenced to identify sequence differences that might account for MD resistance. In total, 2.9+ million polymorphisms were found. Combined with other functional information, we should be able to identify the genes and underlying mechanisms that promote MD resistance, which will lead to accurate and economical methods to rapidly enhance MD resistance in commercial birds.

4. Initial characterization of chicken SCA2, a disease resistance gene in chicken. The vast majority of genes in any species have unknown function. Previously, we identified chicken SCA2 as a gene that confer genetic resistance to Marek’s disease (MD), a T cell lymphoma caused by the Marek’s disease virus (MDV). However, the chicken SCA2 protein has not been characterized, especially with respect to MDV infection. We showed that SCA2 is developmental regulated and expressed in a number of tissues. Most interestingly, MDV US10 protein interacts with and appears to alter the subcellular localization of SCA2. This fundamental knowledge of how MDV infection leads to tumors may aid in better vaccines or other control measures for MD.

5. Evaluation of genetic resistance in chicken on avian influenza disease incidence. Avian influenza is an economically important virus infection that can cause severe economic loss for the poultry industry. The research presented in this report shows that identified differences in genes involved in the chicken's immune respone can affect the resistance or susceptibility to avian influenza. Although these differences in the identifiable chickens immune response genes affect the resistance or susceptibility to avian influenza, this study also shows that there are other unidentified immune response genes as, or more important, than the genes tested in resistance or susceptibility to avian influenza.

6. Experimental method developed to mimic evolution of Marek’s disease virus (MDV). The evolving nature of the Marek’s disease virus (MDV) toward increased virulence is under investigation using characterized strains of MDV and defined chicken lines. The ability to select for increased virulence in a controlled laboratory setting will aid in determining the molecular nature of increased MDV virulence and the influence of the resistant or susceptible characteristics of the defined chicken lines on virus evolution.

7. Identification of a Marek’s disease virus (MDV) protein that helps the virus evade the chicken immune response. Understanding the ability of the Marek’s disease virus (MDV) to evade the chicken’s immune response took a step forward by the discovery that a virus protein (MDV012) can alter virus recognition by the immune system. This virus protein that inhibits the chicken’s immune systems recognition of the viral infection will provide insight into the design of vaccines that stimulate a more potent immune response to MDV.

8. DNA fingerprinting of unique chicken lines developed and maintained at the USDA-ARS, Avian Disease and Oncology Laboratory (ADOL). For 70+ years, ADOL has developed and maintained over 40 unique genetic lines of chickens, which serve scientists at ADOL as well as other non-USDA institutes and universities for research needs. Many of these chicken lines have been DNA fingerprinted with 3K and 60K genetic marker panels. Genetic characterization of the lines enhances the power of scientists using these lines to solve problems facing the poultry industry, especially with respect to infectious disease and animal welfare. Ultimately, US consumers will benefit from this ongoing increase in knowledge in the form of more productive and healthier chickens.

9. Fine-mapping of genes conferring genetic resistance to Marek’s disease (MD). MD is an avian herpesvirus-induced disease, which continues to pose a real threat to the prosperity of the poultry industry. Although MD has been controlled by vaccines since 1970s, the importance of host genetic resistance to MD in fighting against MD is widely recognized. A special chicken population was generated from experimental chicken lines that differ in genetic resistance to MD. In theory, this special population should be more powerful to map and identify genes that confer resistance to MD. Thus far, the population has been generated and characterized with respect to MD incidence, and genetic analyses are proceeding as planned. Once completed, findings from this study should significantly advance the basic understanding of genetic resistance to MD and improve genetic resistance in poultry through molecular selection and breeding.

10. Evaluation of host (chicken) genetics effect on vaccine efficacy. Since their invention, vaccines have proven to be the most effective and economical method to combat infectious diseases in human as well as in livestock. Efforts to improve vaccine protective efficiency have continued and expanded. Host genetics differences were investigated for the influence on Marek’s disease (MD) vaccine efficacy using unique genetic lines of chickens. Our data suggests that host genetics is one of the important factors that influence MD vaccine protection efficiency. This finding should inspire full consideration of host genomics specificity in design, selection, and application of vaccines to maximize vaccine efficiency against infectious diseases.

Review Publications
Muir, W.M., Wong, G., Zhang, Y., Wang, J., Groenen, M., Crooijmans, R., Megens, H., Zhang, H., Okimoto, R., Vereijken, A., Jungerius, A., Albers, G., Taylor Lawley, C., Delany, M.E., Maceachern, S.A., Cheng, H.H. 2008. Genome-Wide Assessment of Worldwide Chicken SNP Genetic Diversity Indicates Significant Absence of Rare Alleles in Commercial Breeds. Proceedings of the National Academy of Sciences. 105(45):17312-17317.

Heifetz, E.M., Fulton, J.E., O'Sullivan, N.P., Arthur, J.A., Cheng, H.H., Wang, J., Soller, M., Dekkers, J.C. 2009. Mapping QTL Affecting Resistance to Marek's Disease in an F6 Advanced Intercross Population of Commercial Layer Chickens. Biomed Central (BMC) Genomics. Available:

Sherman, M.A., Goto, R.M., Moore, R.E., Hunt, H.D., Lee, T.D., Miller, M.M. 2008. Mass Spectral Data for 64 Eluted Peptides and Structural Modeling Define Peptide Binding Preferences for Class I Alleles in Two Chicken MHC-B Haplotypes Associated with Opposite Responses to Marek's Disease. Immunogenetics. 60(9):485-555.

Cheng, H., Niikura, M., Kim, T., Mao, W., MacLea, K., Hunt, H., Dodgson, J., Burnside, J., Morgan, R., Ouyang, M., Lamont, S., Dekkers, J., Fulton, J., Soller, M., Muir, W. 2008. Using integrative genomics to elucidate genetic resistance to Marek's disease in chicken. In: Pinard M-H, Gay, C., Pastoret, P-P, Dodet, B., editors. Integrative Genomics for Disease Resistance: Animal Genomics for Animal Health. Developments in Biologicals. Basel, Switzerland: Karger. p. 365-372.

Zhang, H., Bacon, L.D., Fadly, A.M. 2008. Development of an Endogenous Virus-Free Line of Chickens Susceptible to All Subgroups of Avian Leukosis Virus. Avian Diseases. 52(3):412-418.

Groenen, M.A., Wahlberg, P., Foglio, M., Cheng, H.H., Megens, H.J., Crooijmans, R.P., Besnier, F., Lathrop, M., Muir, W.M., Wong, G.K., Gut, I., Andersson, L. 2009. A High-Density SNP-Based Linkage Map of the Chicken Genome Reveals Sequence Features Correlated With Recombination Rate. Genome Research. 19(3):510-519.

Cheng, H.H. 2008. Integrating genomics to understand the Marek's disease virus-chicken host-pathogen interaction. In: Gustafson, P., Stacey, G., Taylor, J., editors. Genomics of Disease. Stadler Genetics Symposium. New York, NY: Springer. p. 115-126.

Yu, Y., Zhang, H.M., Byerly, M.S., Bacon, L.D., Porter, T.E., Liu, G.E., Song, J. 2009. Alternative Splicing Variants and DNA Methylation Status of BDNF in Inbred Chicken Lines. Brain Research. 1269(7):1-10.

Yu, Y., Zhang, H., Tian, F., Zhang, W., Fang, H., Song, J. 2008. An Integrated Epigenetic and Genetic Analysis of DNA Methyltransferase Genes (DNMTs) in Tumor Resistant and Susceptible Chicken Lines. PLoS One. 3(7):1-13.

Chen, M., Payne, W.S., Dunn, J.R., Chang, S., Zhang, H.M., Hunt, H.D., Dodgson, J.B. 2009. Retroviral Delivery of RNA Interference Against Marek's Disease Virus In Vivo. Poultry Science. 88(7):1373-1380.

Last Modified: 06/23/2017
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