2011 Annual Report 1a.Objectives (from AD-416) Marek’s disease virus (MDV), unlike most other viruses, is highly cell-associated and consequently, exists as population rather due to the inherent process of mutations. This makes it very difficult to determine the relationship between genetic variation and phenotypic variation. With the advent of molecular clones of MDV-BAC clones and genomic technologies, we can now better address how specific genetic changes influence virus virulence, vaccine efficacy, etc. Specifically, we would like to know during cell passage, are there preferred sites that are required for MDV attenuation. The proposed experiments will guide scientists on how to molecular characterize MDV field strains, what changes occur during cell passage attenuation and vaccine production, and other fundamental knowledge. 1b.Approach (from AD-416) Our virulent BAC cloned MDV genome that generates a fully virulent virus will be passed in cell culture, which is known to attenuate the virus. At every 10 passages, the viral population will be used to challenge chickens to determine the amount of MD incidence. This will continue until the viral population is completely avirulent. Preceeding populations will have their viral genome purified and sequenced using next generation sequencers (e.g., Illumina GA or ABI SOLiD) to identify polymorphisms in the genome as well as the allele frequency. In addition, RNAs from the same sequenced populations will be sequenced, which when combined with the genomic sequence information, will confirm polymorphisms and reveal changes in viral gene transcription pattern. Following analysis, key genetic changes will be introduced into the virulent viral genome to address whether the polymorphisms do promote attenuation. 3.Progress Report This project is directly linked to Specific Cooperative Agreement 3635-32000-015-12S titled “Identification, Characterization, and Validation of Genetic Mutations Incurred During in Vitro Attenuation of Marek’s Disease Virus.” This year, we successfully generated viruses that were attenuated, which demonstrates that loss of virulence is due to new mutations rather than selection for pre-existing attenuated Marek's disease viruses that are better adapted for growth in cultured cells. Complete DNA and RNA sequences of the 3 replicates of attenuated viruses and the original parental strain has revealed several candidate genes that may account for attenuation. Of particular interest is ICP4, an immediate early transcription factor, which shows multiple mutations including those that alter the amino acid composition of this protein. Efforts are underway to make recombinant viruses with targeted alterations to validate that these mutations affect virulence.