Location: Avian Disease and Oncology Laboratory
Title: Progress on Identifying Specific Genetic Changes in the Marek's Disease Virus Genome Associated With Attenuation Induced by Repeated In Vitro Passages Authors
|Hildebrandt, Evin -|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 1, 2010
Publication Date: October 18, 2010
Citation: Hildebrandt, E., Silva, R.F., Cheng, H.H. 2010. Progress on identifying specific genetic changes in the Marek's disease virus genome associated with attenuation induced by repeated in vitro passages [abstract]. In: 5th International Workshop on the Molecular Pathogenesis of Marek's Disease Virus and 1st Symposium on Avian Herpesviruses, October 17-20, 2010, Athens, Georgia, p. 65. Technical Abstract: Marek’s disease (MD) vaccines have been successfully used for decades to control tumor formation induced by Marek’s disease virus (MDV), the causative agent and an oncogenic alphaherpesvirus. However, there is still a need for improved MD vaccines as none are sterilizing, and unpredictable vaccine breaks continue to occur spontaneously in the field. MD vaccines are often generated by attenuating virulent MDV strains through repeated passage in vitro. While repeated in vitro passages is a proven method to attenuate MDV, the process is imprecise and, more importantly, there is no guarantee that the resulting attenuated strain will be equal, much less surpass, vaccines already in use. In order to rationally produce more effective MD vaccines, the ideal solution would be to engineer MDV strains with specific changes that result in improved vaccinal protection. The major goal of this project is to determine the precise genetic changes during passage of MDV in vitro that result in attenuation. To facilitate the ability to identify genetic changes, a homogenous MDV strain with a single and known genome sequence was generated from a virulent Md5 BAC clone. Triplicate replicates of the BAC-derived virus and the parental Md5 strain are being repeatedly passaged in vitro. Currently, we are up to 80 passages with viral stocks saved every 10 passages. Studies are underway to evaluate virulence, which will be followed by next generation sequencing of the attenuated strains at the lowest passage number. Sequence analysis will enable us to identify and quantify polymorphisms, and these polymorphisms will be further quantified in the earlier passages to monitor the kinetics of specific sequence changes. To associate virulence with allele abundance in mixed populations, defined mixtures of virulent and avirulent MDVs are being used in bird challenges to provide calibration points on the percent virulent genome associated with MD incidence and related traits. Ultimately, the candidate genes and mutations will be verified via recombineering to see if they alter in vivo virulence. The most current results will be presented.