2007 Annual Report 1a.Objectives (from AD-416) Identify genetic predictors of Marek's disease virus (MDV) virulence. Identify host-viral genetic determinants that control avian tumor virus pathgenicity and shedding. Elucidate the genetic determinants that modulate MDV interactions within the avian immune system. Elucidate host-viral interactions that drive the evolution of new virulent strains of avian tumor viruses. Discover safe and highly effective vaccine platforms that convey protection against emerging MDV strains. 1b.Approach (from AD-416) Avian tumor viruses of economic importance include:. 1)Marek’s disease virus (MDV), a herpesvirus that induces a lymphoproliferative disease of chickens that, in the absence of effective control measures, is capable of causing devastating losses in commercial layer and broiler flocks; and 2) avian retroviruses, namely avian leukosis virus (ALV) and reticuloendotheliosis virus (REV), both are associated with neoplastic diseases and other production problems in poultry. Also, both ALV and REV are potential contaminants of live-virus vaccines of poultry. Critical needs are:. 1)better MDV vaccines to protect against the current and next generation of virulent field strains of MDV; and. 2)a long-term strategy designed to reduce the ongoing emergence of new virulent MDV, and creation of recombinant ALVs through multiple barriers or reduction in viral load and shedding. The primary emphasis will be on molecular approaches to better understand which viral genes are important for immunopathogenesis and shedding of MDV. Parallel studies will monitor the virulence of field strains of MDV and ALV. Studies are also aimed at characterization of new virus isolates and on improving assays for their detection; additional efforts will be devoted to better understand MDV immunity. The project also emphasizes studies on:. 1)elucidating factors involved in creation of recombinant ALVs; and. 2)determining whether REV genome insertion into MDV and fowlpox virus influences transmission and epidemiology of REV. The end product will be a better understanding of viral gene function, virus-host interactions and the development of materials and improved methodology for control of avian tumor viruses. 4.Accomplishments A. Development of semi-congenic chicks to study the role of endogenous avian leukosis virus, TVB Locus, in Marek's disease susceptibility. Chickens from two specialized USDA-ARS-ADOL experimental lines, line-0 and line 0.44-EV21, are being used to produce semi-congenic chicks, which will fall into four groups of TVB genotype and presence or absence of complete endogenous avian leukosis virus (ALV) combinations. These chicks will be used to assess influence of host genotype and endogenous ALV on conferring Marek’s disease (MD) susceptibility. This phase of research is proceeding as planned. Infection with MDV will commence once the chicks are available. The development of such lines will allow us to continue with phase 2 of the experiment, infection with MDV. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses). B. Generation of a Marek's disease virus (MDV) lacking the oncogene named Meq. Using cosmid clones named A6 and SN5, both copies of Meq gene of MDV were deleted by the RecA-assisted restriction endonuclease (RARE) cleavage method. This information is significant, as it identified the Meq deleted MDV mutant as a potential vaccine against MD. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses). C. Analysis of chicken cytokines and other immune related proteins. Using Real-Time PCR analysis of chicken cytokines and other immune related proteins revealed that a T helper 1 type of immune activity that is so critical for the induction of a successful host antiviral immune response does not predominate in very virulent plus MDV-infected chickens. The expression pattern of interleukin (IL)-4, IL-10, and IL13 in the spleen tissues of infected chickens indicates that MDV induces predominantly not a cell-mediated immune response but a humoral type of immunity. This observation is critical in understanding the molecular mechanism of innate and adaptive immunity to MD and the critical role of cytokines in orchestrating immune responses. Using cytokines as genetic adjuvant would be one logical step toward modulating the immune reaction to a vigorous antiviral T helper 1 immune response. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses). D. Development of PCR tests to diagnose avian leukosis and Marek's disease viruses. We developed a PCR that is avian leukosis virus (ALV) subgroup specific and used the PCR to confirm that a contaminant in a Marek’s disease (MD) vaccine was a sub-group A ALV. We also developed a PCR to detect the Rispens strain of MD virus (MDV) in the presence of other serotype 1 MDVs. The development of these diagnostic tests is essential for accurate diagnosis of ALV and MDV. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses). E. Surveillance of field flocks for Marek's disease and avian leukosis viruses. In collaboration with poultry breeders and growers, we continue to monitor field flocks for Marek’s disease virus (MDV) and avian leukosis virus (ALV) for unusual pathogenicity. Samples from commercial layer flocks thought to have a higher incidence of Marek’s disease (MD) were submitted by the University of Pennsylvania. Preliminary results indicated the isolation of pathogenic serotype 1 MD virus (MDV) from commercial layers. Pure stocks of the isolated pathogenic serotype 1 MDV obtained from commercial layer flocks are currently being propagated for future pathotyping studies. This information will add to our knowledge on the pathotype of MDV in the field. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses). F. Cloning of two chicken cytokins. We have successfully cloned two cytokines into plasmids. These cloned cytokines will be inserted into recombinant Marek’s disease (MD) vaccines to improve the efficacy of the vaccine. The information will lead to the development of more effective MDV vaccines. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses). G. Use of a new chicken line to study recombinant avian Leukosis virus (ALV). Our new chicken line named RFS, a line that is susceptible to all subgroups of ALV and lack all endogenous ALV genes is being used to produce fertile eggs for preparation of chicken embryo fibroblasts (CEFs) for use in intial experiments to study conditions that promote recombination of ALV. The availability of these chicken embryos from RFS line will allow us to continue with our studies in recombinant ALV. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses). H. Use of Meq-deleted recombinant rMd5 strain of Marek’s disease virus (MDV) as a vaccine. Preliminary results from field and laboratory studies suggest that this Meq-deleted rMd5 strain of MDV is superior to the best of currently used commercial MDV vaccines in the field. The infromation has led to the development of several material transfer agreements with various vaccine manufacturers for further evaluation as a commercial vaccine. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses). I. Use of Bacterial Artificial Chromosome (BAC) to delete the oncogene termed Meq from Marek's disease virus. One copy of the (MDV) gene, Meq was deleted. Experiments to delete the second copy of Meq in Md5 BAC are ongoing. The potential use of Md BAC with deleted two copies of Meq as vaccine will be studied. This information is significant, as it indicated that BAC technology can be used to delete MDV genes; it is a much more effective technolgy to produce vaccines. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses). 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of new CRADAs and MTAs 2 Number of active CRADAs and MTAs 6