2008 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. Brief descriptions on selected areas are given below. For more details, see Question 4 and the 14 sibling projects. Genetic maps provide the foundation to identifying genes, and the framework for the whole genome sequence. The utility of a genetic map is influenced by the number (saturation) and types of marker employed. Last year, ~13,000 single nucleotide polymorphisms (SNPs), the genetic marker most easily typed by machines, were screened that resulted in the generation of a new consensus map that includes 8600+ SNPs and 667 other markers. Chicken stem cell antigen 2 (SCA2) was previously identified as a Marek’s disease (MD) resistance gene in part due to its direct protein interaction with Marek’s disease virus (MDV) US10. To further confirm and characterize this interaction, a specific antibody was developed for SCA2. Our studies indicate SCA2 is expressed in many chicken tissues and located on the cell surface with a GPI anchor. To gain understanding on the contribution of non-MHC genomic variation to MD genetic resistance and vaccine efficacy, a series of 19 specialized chicken lines were examined by a viral challenge assay with or without MD vaccines. These studies have shed light on the impact of non-MHC regions for MD resistance, MD vaccine protection efficiency, and provide chicken lines to further explore the molecular basis of MD genetic resistance and vaccine control. Cells infected with MDV show greatly enhanced expression of MHC class II, a protein that presents foreign antigens to the chicken immune system, on the cell surface. Using our infectious clones that contain the entire MDV genome, genes that encode proteins previously identified as interacting with components of MHC class II were deleted. It was determined that these MDV genes are necessary for the novel upregulation of MHC class II in MDV-infected cells. Our preliminary studies suggest that these MDV genes influence MD incidence in birds that are naturally infected with MDV. Evolution of MDV to more virulent forms after 8 in vivo passages was tested. In certain pools of passaged lymphocytes (white blood cells) infected with MDV, we identified a “transplantable” tumor line that caused death of animals within 2 weeks after infection of MD vaccinated birds. Thus, vaccination with the most current and effective MD vaccine does not prevent transplantable tumors. The virus re-isolated after 8 passages through birds was slightly more virulent than the starting material. It remains to be determined if this “increase” in virulence is statistically significant. The information and materials produced directly related to NP101 Component 1 as we have: (1) develop genome-enabling tools and reagents for the chicken, e.g., high-density genetic maps, (2) identified a number of functional genes and their interactions both within the chicken and to MDV, (3) quantified the extent of remaining genetic diversity within commercial poultry lines, and (4) have initiated efforts to implement genome-wide marker-assisted selection (GWMAS) in poultry. 4.Accomplishments 1. RNA interference to inhibit viral disease in chickens. Modern vaccines have reduced productivity loss to viral diseases, however, many viral diseases continue to decrease animal productivity and welfare. Additional tools to complement vaccinal control methods could aid in further reducing the negative effects of viral disease. Recently, a system known as RNA interference or RNAi has been developed that reduces the expression of specific genes. Taking advantage of this method, we have adapted this technology to reduce the severity of viral infections in chickens by targeting virus genes. The feasibility of this approach was shown in live birds where Marek’s disease virus replication and pathogenesis has been reduced. This method has the potential to inhibit any infectious disease and may offer a valuable tool to control disease. This accomplishment addresses NP 101 Component 1 (Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources) and Problem Statement 1A (Develop Genome-Enabling Tools and Reagents). 2. Improved chicken genetic map. 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. Molecular genetics the marker-assisted genetic using DNA-based technologies is an attractive solution as it allows increased selection intensity, increased accuracy of selection, and maintains or integrates new genetic variation. Contingent on this is the availability of a high quality genome sequence and accompanying genetic markers to tag genes of interest. In the past year, ~13,000 genetic markers that can be rapidly and economically typed were screened on our existing mapping populations that resulted in a new chicken genetic map that includes 9,000+ markers. This valuable tool greatly enhances our ability to identify genes of economic importance and improves the chicken genome sequence assembly. This accomplishment addresses NP 101 Component 1 (Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources) and Problem Statement 1A (Develop Genome-Enabling Tools and Reagents). 3. A fast method to identify genes underlying complex traits such as disease resistance. Identification of individual genes that control complex traits such as disease resistance is difficult. One possible solution is to monitor allele-specific expression (ASE), i.e., individual expression of each of the two alleles found for each gene. If found, this indicates there is a genetic element present for the gene of interest. In the past year, we have identified a list of genes that we wish to screen for sequence variants, which are required to tract individual alleles. Also, a program has been designed to amplify each one of these genes, which we will use to identify sequence variants between the two lines, the only requirement for us to follow alleles of each gene. If successful, this approach will greatly enhance our ability to identify genes of economic importance. This accomplishment addresses NP 101 Component 1 (Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources) and Problem Statement 1B (Identify Functional Genes and Their Interactions). 4. Understanding the role and impact of non-MHC genomic variation on Marek’s disease (MD) resistance and vaccine efficacy. Genetic resistance offers alternative control methods for MD besides vaccination. Prior to its implementation, the causative genes need to be found in the chicken genome. Data from our experiments using specialized chicken lines strongly suggest there are regions with genes that confer different levels of MD resistance and/or vaccinal protection. These lines will greatly aid in our efforts to understanding and define MD resistance genes. These accomplishments addresses NP 101 Component 1 (Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources) and Problem Statement 1B (Identify Functional Genes and Their Interactions). 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings 2 Review Publications Muir, W.M., Wong, G.K., Zhang, Y., Wang, J., Groenen, M.A., Crooijmans, R.P., Megens, H.J., Zhang, H.M., McKay, J.C., McLeod, S., Okimoto, R., Fulton, J.E., Settar, P., O'Sullivan, N.P., Vereijken, A., Jungerius-Rattink, A., Albers, G.A., Lawley, C.T., Delany, M.E., Cheng, H.H. 2008. Review of the initial validation and characterization of a 3K chicken SNP array. World Poultry Science Journal. 64:219-225. Yul, Y., Zhang, H.M., Tian, F., Bacon, L.D., Zhang, Y., Zhang, W., Song, J. 2008. Quantitative evaluation of DNA methylation patterns for ALVEs and TVB genes in a neoplastic disease susceptible and resistant chicken model. PLoS One. 3(3):e1731. Cogburn, L.A., Porter, T.E., Duclos, M.J., Simon, J., Burgess, S.C., Zhu, J., Cheng, H.H., Dodgson, J.B., Burnside, J. 2007. Functional genomics of the chicken - a model organism. Poultry Science. 86:2059-2094. Mao, W., Niikura, M., Silva, R.F., Cheng, H.H. 2008. Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL41 gene via an in vitro competition assay. Journal of Virological Methods. 148:125-131.