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United States Department of Agriculture

Agricultural Research Service

Research Project: IDENTIFICATION, CHARACTERIZATION, AND VALIDATION OF GENETIC MUTATIONS INCURRED DURING IN VITRO ATTENUATION OF MAREK’S DISEASE VIRUS
2010 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 linked to research project 3635-32000-015-09R “Identification, Characterization, and Validation of Genetic Mutations Incurred During In Vitro Attenuation of Marek’s Disease Virus.” Marek's disease (MD) is one of the most serious chronic threats to the U.S. poultry industry due to recurring yet unpredictable vaccine breaks. Genetic mutations in Marek's disease virus (MDV), the causal pathogen, play a pivotal role in both the evolution of field strains with higher virulence and in vitro (cell culture) attenuation, the process used to make traditional vaccines. Unfortunately, little is known about naturally-occurring MDV mutations and their influences on virulence. This year, starting with our infectious BAC (plasmid) clone containing the entire MDV genome, we generated the virus and passed triplicate replications in chicken cells. At cell passage 60 and 70, all three replicates became completly attenuated based on challenge experiments in highly susceptible chickens. This indicates and confirms past results to show that the viral genome picks up random mutations that will eventually attenuate the virus. These attenuated populations form the basis for future studies to identify all the changes in the viral genome. This information will help promote the rational design of superior MD vaccines, provide molecular markers of MDV pathotypes, and aid in the prediction of MDV evolution in the field. This project is monitored by monthly e-mail and telephone calls between the two parties and, when possible, direct interactions at scientific meetings.


Last Modified: 12/17/2014
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