Technical Abstract: The Marek’s disease virus (MDV) genome encodes about 110 open reading frames (ORFs). Many of these ORFs are annotated based purely on homology to other herpesvirus genes, thus, direct experiments are needed to verify the gene products, especially the hypothetical or MDV-specific ORFs, and characterize their biological function, particularly with respect to pathogenicity in chickens. Previously, a comprehensive two hybrid assay screen revealed nine specific chicken-MDV protein-protein interactions. To characterize the role of hypothetical MDV proteins R-LORF10 and LORF4, which were shown to interact with major histocompatibility complex (MHC) class II beta chain and Ii (invariant or gamma) chain, respectively, recombinant MDVs derived from virulent MDV-BAC clone rMd5-B40 were generated. Recombinant MDV rMd5deltaR-LORF10 lacked part of the promoter and the first 17 amino acids in both copies of R-LORF10, and rMd5mLORF4 had point mutations in LORF4 that disrupted the start codon and introduced a premature stop codon without altering the amino acid sequence of overlapping ORF UL1, which encodes glycoprotein L (gL). Mutations in either R-LORF10 or LORF4 did not prevent MDV reconstitution from modified MDV-BACs nor significantly alter virus growth rate in vitro. However, MDV generated from rMd5deltaR-LORF10 had reduced virulence compared to the parental MDV. Surprisingly, MDV with the LORF4 mutations had significantly higher overall MD incidence as measured by mortality, tumor production, and MD symptoms in infected chickens. These results indicate R-LORF10 and LORF4 encode real products and are involved in MDV virulence although their mechanisms, especially with respect to modulation of MHC class II cell surface expression, are not clearly understood.