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.
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. This project is monitored by monthly e-mail and telephone calls between the two collaborating parties and, when possible, direct interactions at scientific meetings.