2012 Annual Report 1a.Objectives (from AD-416): As described in USDA AFRI-funded grant where Dr. Niikura is a co-PI, we will: 1. Identify and correlate specific genetic and functional changes in the MDV genome that occur during the in vitro attenuation process with virulence. 2. Confirmation of the relevance of the data obtained in objective 1 to the attenuation process. 1b.Approach (from AD-416): With respect to Objective 1, we will: 1. Serially passed BAC-cloned Marek’s disease virus (MDV) Md5 strain in triplicate and test for virulence. 2. Characterize genetic changes in the MDV genome using next generation sequencing, and correlate the allele frequency results with the level of virulence. 3. Characterize sequence changes that occur during the attention process using next generation sequencing, and correlate the expression differences with the level of virulence. 4. Determine SNP allele frequencies in MD vaccines. With respect to Objective 2, we will: 1. Validate changes identified in objective 1 but introducing defined mutations in our MDV-BAC clone. 3.Progress Report: This project is directly linked to project 3635-32000-016-02R titled “Identification and Characterization, and Validation of Genetic Mutations Incurred During in Vitro Attenuation.” This year, we molecularly characterized the 3 replicates of attenuated MDVs as well as the parental virulent MDV. Depending on the replicate, 41 to 95 SNPs were identified that were present in at least 2% of the viral population. Genes with non-synonymous mutations in all attenuated populations were UL26 and ICP4. Other promising candidates based on their kinetics of emergence over the cell passages include UL5, UL42, UL46, and MDV011. Defined recombinant MDVs have been constructed for many of these mutations and bird challenge experiments are ongoing to determine if these polymorphisms alter viral pathogenicity.