Identifying the Genetic Basis of Attenuation in Marek's Disease Virus via Experimental Evolution

Marek’s disease virus (MDV) is an oncogenic alphaherpesvirus of chickens that induces lymphoid tumors. Control of this disease, which causes $2+ billion in annual losses worldwide, is primarily through vaccination. Presently, the most efficacious vaccines are attenuated MDV strains generated by repeated blind cell passages. While these vaccines prevent tumor formation, they are non-sterilizing and likely promote the emergence of more virulent field strains. The goal of this project is to identify the molecular basis of in vitro attenuation. To identify polymorphisms associated with attenuation, virulent MDV generated from a BAC clone was serially passed in cells in triplicate. Birds were challenged with passed stocks to determine the lowest passage at which attenuation occurred, then both viral DNA and RNA was subjected to Illumina sequencing. Comparing attenuated replicates versus virulent parental virus identified 40-100 SNPs. Candidate SNPs whose frequency exceeded 20% were selected for targeted resequencing to track their frequency every 10 passages. High frequency mutations whose frequency increased between the passages in which virulence decreased were picked as top candidate mutations to introduce into recombinant viruses. Five of the top candidate SNPs within genes UL5, UL42, UL46, and MDV012 have been incorporated into the parental BAC clone to generate defined recombinant MDVs. In bird trials, four recombinant viruses retained virulence comparable to the parental virus, however, the fifth point mutation within UL5 caused a dramatic decrease (90+%) in disease incidences.