Marek's disease (MD) in chickens is caused by the alphaherpesvirus MD virus (MDV) and is characterized by the development of lymphoblastoid tumors in multiple organs. The recent identification and cloning of RLORF4 and the finding that four of six attenuated strains of MDV contained deletions within RLORF4 suggested that it is involved in the attenuation process of MDV. To assess the role of RLORF4 in MD pathogenesis, its coding sequence was deleted in the pRB-1B bacterial artificial chromosome clone. Additionally, RLORF5a was deleted separately to examine its importance for oncogenesis. The sizes of plaques produced by MDV reconstituted from pRB-1BdeltaRLORF5a (rRB-1BdeltaRLORF5a) were similar to those produced by the parental pRB-1B virus (rRB-1B). In contrast, virus reconstituted from pRB-1BDeltaRLORF4 (rRB-1BdeltaRLORF4) produced significantly larger plaques. Replication of the latter virus in cultured cells was higher than that of rRB-1B or rRB-1BdeltaRLORF5a using quantitative PCR (qPCR) assays. In vivo, both deletion mutants and rRB-1B replicated at comparable levels at 4, 7, and 10 days postinoculation (p.i.), as determined by virus isolation and qPCR assays. At 14 days p.i., the number of PFU of virus isolated from chickens infected with rRB-1BdeltaRLORF4 was comparable to that from chickens infected with highly attenuated RB-1B and significantly lower than that from rRB-1B-infected birds. The number of tumors and kinetics of tumor production in chickens infected with rRB-1BdeltaRLORF5a were similar to those of P2a chickens infected with rRB-1B. In stark contrast, none of the chickens inoculated with rRB-1BdeltaRLORF4 died up to 13 weeks p.i.; however, two chickens had tumors at the termination of the experiment. The data indicate that RLORF4 is involved in attenuation of MDV, although the function of RLORF4 is still unknown.
