Characterization of integrated Marek's disease virus genomes supports a model of integration by homology-directed recombination and telomere-loop-driven excision.

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that readily infects chickens and some other poultry, triggering complex disease phenotypes, including paralysis, immunosuppression, and lymphoma leading to death. MDV infection is partially controlled by extensive global vaccination programs but nevertheless, it imposes a significant financial burden on commercial poultry farming. Following infection, the MDV genome integrates into host telomeres, which is associated with the virus entering a latent state. The mechanism of integration remains poorly understood but it is contemporaneous with cellular transformation and lymphoma formation and therefore requires investigation. Here we have developed droplet digital PCR assays to quantify different regions of the MDV genome. We have also used long-range PCR and single telomere amplification to establish the organization and relative orientation of MDV genome regions in integrated MDV (iMDV). These analyses show that following integration, the MDV genome is oriented with the unique short region (U S ) internal to the unique long region (U L ) and that an iMDV-associated telomere forms at the variable repeat array (mTMR) in a terminal a-like sequence. The data also reveal a very wide range of MDV copy numbers in cell lines, including unexpectedly, additional copies of specific MDV genome regions. IMPORTANCE Marek's disease virus (MDV) is a ubiquitous chicken pathogen that inflicts a large economic burden on the poultry industry, despite worldwide vaccination programs. MDV is only partially controlled by available vaccines, and the virus retains the ability to replicate and spread between vaccinated birds. Following an initial infection, MDV enters a latent state and integrates into host telomeres and this may be a prerequisite for malignant transformation, which is usually fatal. To understand the mechanism that underlies the dynamic relationship between integrated-latent and reactivated MDV, we have characterized integrated MDV (iMDV) genomes and their associated telomeres. This revealed a single orientation among iMDV genomes and the loss of some terminal sequences that is consistent with integration by homology-directed recombination and excision via a telomere-loop-mediated process.