Genetic homogeneity of a test population is essential to precisely associate a viral genome sequence and its phenotype at the nucleotide level. However, homogeneity is not easy to achieve for Marek's disease virus (MDV) due to its strictly cell-associated replication. To address this problem, two virulent infectious bacterial artificial chromosome (BAC) clones of MDV were generated from an MDV genome previously cloned as five overlapping cosmids. The Md5SN5BAC clone has the BAC vector inserted between the 3' ends of UL3 and UL4, such that no known ORFs should be disrupted. The BAC vector is flanked by loxP sites, so that it can be deleted from the viral genome by transfecting Md5SN5BAC into a newly developed chicken cell line that constitutively expresses Cre recombinase. The Md5B40BAC clone has the BAC vector replacing a portion of US2, a location similar to that used by other groups to construct MDV-BAC clones. Although both BACs were capable of producing infectious virulent MDV when inoculated into susceptible chickens, Md5B40BAC-derived viruses showed somewhat better replication in vivo and higher virulence. Removal of the BAC vector in Md5SN5BAC-derived viruses had no influence on virulence. Interestingly, when genetically homogeneous virulent MDV generated from Md5B40BAC was mixed with avirulent virus, the overall virulence of the mixed population was noticeably compromised, which emphasizes the importance of MDV population complexity in pathogenesis.

Marek's disease (MD) is a T-cell lymphoma of chickens caused by the oncogenic Marek's disease virus (MDV). MD is primarily controlled by live-attenuated vaccines generated by repeated in vitro serial passage. Previous efforts to characterize attenuated MDVs identified numerous mutations, particularly a convergence of high-frequency mutations around amino acids 60–63 within ICP4 (RS1), therefore, ICP4 was considered a candidate gene deserving further characterization. Recombinant MDVs were generated containing a single Q63H mutation or double Q63H+S1630P mutations. Despite the repetitive nature of mutations within ICP4, neither recombinant virus decreased virulence, although one mutant reduced in vivo replication and failed to transmit horizontally. Our results indicate that these mutations are insufficient to reduce disease incidence in infected birds, and suggest that variants in ICP4 do not directly alter virulence, but rather may enhance MDV replication rates in vitro, offering an explanation for the widespread occurrence of ICP4 mutations in a variety of attenuated herpesviruses