Characterizing the Molecular Basis of Attenuation of Marek's Disease Virus via In Vitro Serial Passage Identifies De Novo Mutations in the Helicase-Primase Subunit Gene UL5 and Other Candidates Associated with Reduced Virulence

UNLABELLED: Marek's disease (MD) is a lymphoproliferative disease of chickens caused by the oncogenic Gallid herpesvirus 2, commonly known as Marek's disease virus (MDV). MD vaccines, the primary control method, are often generated by repeated in vitro serial passage of this highly cell-associated virus to attenuate virulent MDV strains. To understand the genetic basis of attenuation, we used experimental evolution by serially passing three virulent MDV replicates generated from an infectious bacterial artificial chromosome (BAC) clone. All replicates became completely or highly attenuated, indicating that de novo mutation, and not selection among quasispecies existing in a strain, is the primary driving force for the reduction in virulence. Sequence analysis of the attenuated replicates revealed 41 to 95 single-nucleotide variants (SNVs) at 2% or higher frequency in each population and several candidate genes containing high-frequency, nonsynonymous mutations. Five candidate mutations were incorporated into recombinant viruses to determine their in vivo effect. SNVs within UL42 (DNA polymerase auxiliary subunit) and UL46 (tegument) had no measurable influence, while two independent mutations in LORF2 (a gene of unknown function) improved survival time of birds but did not alter disease incidence. A fifth SNV located within UL5 (helicase-primase subunit) greatly reduced in vivo viral replication, increased survival time of birds, and resulted in only 0 to 11% disease incidence. This study shows that multiple genes, often within pathways involving DNA replication and transcriptional regulation, are involved in de novo attenuation of MDV and provides targets for the rational design of future MD vaccines. IMPORTANCE: Marek's disease virus (MDV) is a very important pathogen in chickens that costs the worldwide poultry industry $1 billion to $2 billion annually. Marek's disease (MD) vaccines, the primary control method, are often produced by passing virulent strains in cell culture until attenuated. To understand this process, we identified all the changes in the viral genome that occurred during repeated cell passage. We find that a single mutation in the UL5 gene, which encodes a viral protein necessary for DNA replication, reduces disease incidence by 90% or more. In addition, other candidate genes were identified. This information should lead to the development of more effective and rationally designed MD vaccines leading to improved animal health and welfare and lower costs to consumers.