Sequencing of hypermutated Marek's Disease Virus (MDV, GaHV-2) genomes derived from populations established by exonuclease deficient polymerase mutants.

Using Marek’s Disease Virus (MDV, GaHV-2) as a model, we have examined the effect of a reduction in DNA polymerase fidelity on herpesvirus genetic variability and pathogenicity. Under certain cell culture conditions, low fidelity mutants were able to escape lethal mutagenesis and showed partial restoration of their proof-reading capacity on a population level. These viruses gave rise to a genetically extremely diverse population that cumulatively regained parental-like fitness in cell culture. Using viruses derived from bacterial artificial chromosomes, we were able to separate genetically diverse viral populations into individual genomes in E. coli. Additionally, we developed a custom target enrichment panel to sequence diverse populations of the avidly cell associated MDV. Animal experiments were conducted with the genetically diverse population obtained from a proof-reading deficient virus, which was shown to be markedly more virulent than the (non-diverse) population of the parental virus. We compared cell culture derived virus populations and clones with virus populations and clones derived from infected animals and found high viral sequence diversity to be associated with a very virulent viral phenotype.