2008 Annual Report 1a.Objectives (from AD-416) 1. Identify genetic predictors of MDV virulence. (SEPRL) (ADOL) 2. Identify host-viral genetic determinants that control avian tumor virus pathogenicity and shedding. (ADOL) (SEPRL) 5. Discover safe and highly effective vaccine platforms that convey protection against emerging Marek’s disease viral strains. (SERPL) (ADOL) 1b.Approach (from AD-416) 1. Identify genetic predictors of MDV virulence. The nucleotide sequences of the long repeat region in the genomes of both attenuated and mildly virulent strains of MDV will be determined using conventional sequencing technologies. Viral DNA will be isolated, subjected to the polymerase chain reaction and sequenced. Whole genome sequences of serial passaged viruses will be determined using 454 Life Sciences technologies. To determine the collection of mutations responsible for attenuation and the multigenic nature of attenuation, virus samples at defined passage intervals will be collected and processed for pathotyping in a bioassay and 454-based sequencing. 2. Identify host-viral genetic determinants that control avian tumor virus pathogenicity and shedding. The expression level of viral genes and their effects on the expression of cellular genes will be examined using Chicken genome microarrays. Two chicken lines (72 and 63) infected with various MDV pathotypes will be used in these studies. Various tissue samples will be collected 5 days post challenge and RNA will be isolated using commercially available reagents. cDNA will then be made for isolated RNA, labeled with fluorophores and hybridized to the microarrays. 5. Discover safe and highly effective vaccine platforms that convey protection against emerging Marek’s disease viral strains. The genomes of currently circulating very virulent plus (vv+) pathotypes will be modified by deletion of virulence genes and incorporation of genes encoding immune modulators (i.e. cytokines, soluble cytokine receptors and microrna constructs) in order to modulate the TH1/TH2 immune response. To achieve this, vv+ genomes will be cloned as a bacterial artificial chromosome and modified using recE/T mutagenesis of E.coli. Recombinants will be tested for loss of pathogenicity in birds. Efficacy of vaccine candidates will then be determined in protection trials. 3.Progress Report Our comparative Marek’s disease virus genomic program has identified virulence factors that are relevant to the goals outlined in the National Program 103 Animal Health Action Plan, Component 2 Genetic and Biological Determinants of Disease Susceptibility, Problem Area 2B Avian Tumor Viruses. In the beginning of Fiscal Year 2008, we determined the nucleotide sequence of the mildly virulent strain (CU-2) of Marek’s disease virus-1 (MDV-1). This strain contained genetic characteristics resembling those of both vaccine and disease–causing strains. We also completed the sequence, annotation and analysis of a second attenuated strain of MDV-1, known as 584Ap80. This strain was attenuated through passage in cultured cells and contained mutations within 18 genes. Mutations in a subset of these genes were previously identified in other lesser virulent strains providing additional evidence that these genes encode virulence factors. Using a functional genomic approach, two MDV-1 genes (UL13 and UL44) were identified that are essential for transmission of the virus from chicken-to-chicken. Understanding the mechanism by which virus sheds will allow us to develop strategies to prevent virulent virus shedding in vaccinated animals, thus reducing the incidence of Marek’s disease in a flock house. During the remainder of Fiscal Year 2008, three comparative genomic projects dominated the work agenda. The first two projects involved the deoxyribonucleic acid (DNA) sequence determination of a commercial MDV-1 vaccine strain (CVI988-699) and an MDV-2 strain that is part of a widely used bivalent Marek’s disease vaccine formulation. The third project involved the sequence determination and comparison of six cell culture passages (10, 30, 40, 60, 80 and 100) of the MDV-1 strain 648A. Analysis of the sequencing data has identified deletions in four regions of the MDV-1 genome, as well as mutations in the origin of replication and two genes of unknown function. All of these genetic changes occurred in late passage (after p60), coinciding with the loss of paralysis and tumor formation. Analysis of the mutations in the genomes at each passage has indicated that MDV-1 exists as a collection of mutated genomes or quasi-species. This is the first report of a herpesvirus existing as a quasi-species. This term is usually reserved for ribonucleic acid (RNA) viruses, not DNA viruses. This discovery elevates MDV-1 genomics to a new level of complexity. Lastly, we completed the sequence analysis of two hypervirulent MDV-1 strains isolated in Europe. These represent the only completed MDV-1 genomes of strains from outside the United States. 4.Accomplishments 1. Sequence analysis of the mildly virulent strain of Marek's disease virus (MDV-1) known as CU-2. Little is known about mutations involved in virulence and attenuation. The CU-2 strain is unique in this regard since it is more virulent than vaccine strains and more attenuated than field isolates. The complete deoxyribonucleic acid (DNA)sequence of this strain was determined and genes were identified that may play a role in its attenuated and virulent characteristics. These results will further our knowledge of genes involved in virulence and allow us to generate more protective vaccines. This accomplishment is in National Program Component 103 Animal Health, Component 2 Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Identify genetic predictors of MDV virulence. 2. The sequence analysis of an attenuated serially-passaged strain of Marek’s disease virus-1 (MDV-1). Serial passage of virulent field isolates results in their attenuation at some point during passage. The changes in genes (i.e. mutations) which result in full attenuation are unknown. The DNA sequence of a serially-passaged virus, which resulted in its attenuation, was determined. Mutations were identified in 18 genes relative to its virulent parental strain. Identification of genes involved in virulence will impact the MDV vaccine industry by indicating which genes can be deleted to produce safer vaccines and which genomic locations can be disrupted so that foreign genes (e.g. cytokines, other chicken pathogen genes) can be inserted to generate improved or bivalent vaccines. This accomplishment is in National Program Component 103 Animal Health, Component 2 Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Identify genetic predictors of MDV virulence. 3. Identification of two Marek’s disease virus-1 (MDV-1) genes(UL13 and UL44) that are essential for chicken-to-chicken spread. Comparative genomics has identified five genes which may play a role in the ability of Marek’s disease virus to spread from bird-to-bird. Using a functional genomic approach in which the five mutated genes were reverted to their wild type genotype, only two (UL13 and UL44) were identified to be essential for transmission. This will have tremendous impact on the vaccine industry, for newer vaccines can be created that will be unable to spread. It is largely suspected that vaccine strains and field isolates recombine to generate new strains. Eliminating one ingredient in this mixing pot could reduce the creation of new virulent field strains. This accomplishment is in National Program Component 103 Animal Health, Component 2 Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Identify genetic predictors of MDV virulence. 4. During infection in cell culture Marek’s disease virus-1 (MDV-1) genomes are not clonal and exist as a collection of mutants or quasi-species. The question remains whether comparative genomics is capable of identifying predictors of virulence shift. By sequencing MDV-1 genomes using newer sequencing technologies we have proven that MDV-1 exists as a collection of mutants similar to RNA viruses. This scientific breakthrough has changed our narrow views on what characteristics of genes (polymorphisms) contribute to virulence in an additive or transdominant fashion. This discovery will impact the scientific community as a whole and allow for an enhanced understanding of the complexity of Marek’s disease viral genomics. This accomplishment is in National Program Component 103 Animal Health, Component 2 Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C: Identify genetic predictors of MDV virulence. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Review Publications Spatz, S.J., Rue, C.A. 2008. Sequence determination of a mildly virulent strain (CU-2) of Gallid herpesvirus type 2 using 454 pyrosequencing. Virus Genes. 36:479-489. Spatz, S.J., Rue, C.A., Schumacher, D., Osterrieder, N. 2008. Clustering of mutations within the inverted repeat regions of a serially-passaged attenuated gallid herpesvirus type 2 strain. Virus Genes. 37:69-80. Smith, K.A., Colvin, C.J., Weber, P.S., Spatz, S.J., Coussens, P.M. 2008. High titer growth of human and avian influenza viruses in an immortalized chick embryo cell line without the need for exogenous proteases. Vaccine. 26:3778-3782.