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Marek's disease (MD) is a highly transmissible, herpesvirus-associated malignancy of chickens and turkeys caused by Marek's disease virus (MDV). MD is currently controlled through the use of nonsterilizing vaccines composed of antigenically related, apathogenic herpesviruses Mardivirus 2 (MDV-2), Meleagrid herpesvirus 1 (herpesvirus of turkeys, HVT), or attenuated MDV-1 strain CVI988 (Rispens). Since the mid-1960s, field strains of MDV have increased in virulence, due, in part, to the widespread use of vaccines since the early 1970s. One mutation that we have identified common to very virulent field strains (vv and vv+MDVs) since the 1990s has been a mutation in the UL1 gene, encoding glycoprotein L (gL). This mutation, a 12-nucleotide (nt) deletion in the signal peptide of gL, has been associated with increased virulence and decreased vaccine protection in the context of challenge with a vv+MDV, strain TK. To determine whether this mutation alone was sufficient to confer increased virulence, we introduced this mutation into the transmission-competent pRB-1B bacterial artificial chromosome (BAC) using two-step, Red-mediated recombination. The resulting mutant, pRB-1BgLdelta, was tested for changes in replication in cell culture using multistep growth curves, plaque size analysis, viral burst analysis, and the ability to compete with the parental virus when co-transfected at different ratios and sequentially passaged. In addition, we examined this mutant for changes in pathogenicity in inoculated and contact-exposed unvaccinated and vaccinated chickens. Our data show minor differences in plaque sizes in cell culture, but no discernible changes in the infection of specific-pathogen-free (SPF) leghorn chickens. We therefore conclude that although this mutation is indeed common to MDV field strains isolated in the eastern United States, it is insufficient to confer increased virulence or loss of vaccine protection previously observed for a vv+MDV strain having this mutation.