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Marek's disease virus (MDV) causes a devastating disease in chickens characterized by the development of lymphoblastoid tumors in multiple organs and is transmitted from the skin of infected chickens. We have previously reported that the U(S)2, U(L)44 (glycoprotein C [gC]), and U(L)13 genes are essential for horizontal transmission of MDV in gain-of-function studies using an a priori spread-deficient virus that was based on an infectious clone from the highly virulent RB-1B virus (pRB-1B). To precisely determine the importance of each individual gene in the process of chicken-to-chicken transmission, we used the transmission-restored clone that readily transmits horizontally and mutated each individual gene in loss-of-function experiments. Two independent U(S)2-negative mutants transmitted horizontally, eliminating U(S)2 as being essential for the process. In contrast, the absence of gC expression or mutating the invariant lysine essential for U(L)13 kinase activity abolished horizontal spread of MDV between chickens.