2007 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 In the first quarter of FY2007 we completed a comparative genomic project involving the sequence determination of the “a-like sequence” from13 strains of GaHV-2.We also obtained the raw sequencing data (from 454 Life Sciences) for the genomes of the attenuated strains of GaHV-2 : CU-2, 584Ap80C, BAC 10 and BAC15. Inconsistencies with the DNA sequences of the two repeat long regions within the sequenced genome of another GaHV-2 strain known as RB-1B-5 (a mutant incapable of spreading bird-to-bird) were resolved during this period and the sequence was submitted to GenBank. During the second quarter of FY2007, three projects dominated the work agenda. These projects involved the determination of the sequence at the ends of the linear form of GaHV-2, determination of the sequence in the GaHV-2 genome required for cleavage of the genomes necessary for mature virus particle formation, and a methodology project involving in vitro amplification of whole viral genome in samples containing a large amount of contaminating host cellular DNA. Progress on projects in the third quarter of FY2007 involved Milestones A-E dealing with CRIS objective 1 and approach 1. This involved the acquisition of viral preps that were generated during a project originally started by our collaborators at ADOL (E. Lansing, MI). The objective of this earlier project was to determine the pathotype (or disease capacity) of serial passaged strains of GaHV-2. My interest in this project involves a comparison between the pathogenesis data and the genetic data I plan on generating by determining the complete nucleotide sequence of the viruses present in the interval-defined samples. To achieve this goal, 8 viral preps were received from ADOL and subjected to a single round of cell culture propagation. Primary cells (Fibroblasts) were isolated from fertilized eggs and used to propagate the viruses in vitro. Methodologies for the isolation of viral DNA from virus particles (e.g. virions) were developed and implemented. These methodologies involved the isolation of viral nucleocapsids, destruction of host DNA using a DNA degradation enzyme (e.g.exonuclease), extraction of viral DNA and removal of small molecular weight DNA using polyethylene glycol precipitation. It is anticipated that the sequencing of these viral samples will be completed in the fourth quarter of FY2007. 4.Accomplishments a. The most significant discovery was the identification and characterization of genetic changes (e.g. polymorphisms) within the “a-like” region spanning the internal repeat long and internal repeat short junction among the 5 pathotypes of Gallid herpesvirus type 2 (GaHV-2). Determining the DNA sequence of this region was extremely difficult due to the presence of numerous repeat elements which are unstable during experimental manipulations. The discovery that attenuated strains (e.g. strains limited in their disease capacity and often used as vaccines) contain deletions in this region that encompass regulatory genes (e.g. RNAi) and their control elements ( e.g. promoter of LAT) will allow for a better understanding of virulence, pathogenesis and lead to the development of improved vaccines. National Program Component 103 Animal Health Problem Statement: Identify genetic predictors of MDV virulence. b. The complete nucleotide sequence of two attenuated strains of GaHV-2 (584Ap80C and CU-2) was determined using revolutionary technology pioneered by 454 Life Sciences. Traditionally the determination of the DNA sequence using Sanger-based technologies has been tedious and expensive. I decided to accelerate this process by using innovative technology based on pyrosequencing at approprimately 1/7th the cost. This endeavor proved fruitful with the sequence completion of two attenuated strains and the identification of numerous mutations that differ in comparison with virulent strains. Bioinformatic analyses of these sequences revealed that both strains contain changes in the meq loci relative to virulent strains. This provided additional evidence that the meq locus is the main players in virulence. National Program Component 103 Animal Health Problem Statement: Identify genetic predictors of MDV virulence. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 3 Number of newspaper articles and other presentations for non-science audiences 2 Review Publications Spatz, S.J., Nair, V. 2007. Comparative full-length sequence analysis of oncogenic and vaccine (Rispens) strains of Marek's disease virus. Journal of General Virology. 88:1080-1096. Spatz, S.J., Silva, R.F. 2007. Sequence determination of variable regions within the genomes of Gallid herpesvirus-2 pathotypes. Archives of Virology. Available: http://www.springerlink.com/content/ku4064776312w861/. Jarosinski, K.W., Margulis, N.G., Kamil, J.P., Spatz, S.J., Nair, V.K., Osterrieder, N. 2007. Horizontal transmission of Marek¿s disease virus requires US2, the UL13 protein kinase, and gC. Journal of Virology. Oct;81(19):10575-87. Spatz, S.J., Silva, R.F. 2007. Polymorphisms in the long repeat regions of oncogenic and attenuated pathotypes of Marek's disease virus 1. Virus Genes. 35(1):41-53.