The overall goal of this project is to understand the mechanistic importance of UL13 protein kinase on viral US10 and cellular LY6E during horizontal transmission. Incorporating the host genetics in our studies is a logical progression of our work to understanding genetic resistance/susceptibility to MD that will facilitate genetic breeding or engineering to develop chickens. The ultimate goal is to 1) reduce shedding of virus and 2) reduce infection of naïve hosts. This strategy will reduce the incidence and the severity of MD, and subsequently reduce the evolutionary pressure current vaccines place on circulating field stains. Our overall goal will be addressed in two major Objectives:Objective 1 is to determine the importance of US10 and phosphorylation on S20 for horizontal transmission. MDV UL13 is dispensable for replication of MDV in vitro and in vivo; however, it is required for chicken-to-chicken transmission. Importantly, UL13 protein kinase activity is required for this mechanism, linking phosphorylation to its essential role in transmission. Using a proteomics approach, we have identified the MDV US10 protein phosphorylated on S20 in MDV-infected feather follicle epithelial (FFE) cells, while this site was unmodified in UL13 kinase mutant-infected FFE cells. Since US10 has been linked to interaction with LY6E and LY6E is involved in late virus entry steps, we hypothesize the requirement of UL13 is due to modification of US10 that allows it to interact with LY6E; thereby, mediating escape from degradation of the nucleocapsid in the endosome of the newly infected cell. Importantly, LY6E is believed to be a MD resistance gene strengthening the role LY6E may play in this model. Here, our objective is to determine whether US10 and specifically, its phosphorylation on S20, is required for transmission. Specific objectives include:Determine that US10 is required for MDV horizontal transmission.Determine that US10 Serine 20 is required for horizontal transmission.Determine that MDV UL13 directly phosphorylates US10 in vitro.Determine that MDV UL13 kinase activity is important for incorporation of US10 in the virion.Objective 2 is to determine the role LY6E plays in horizontal transmission and genetic resistance to MDV. Our overall hypothesis involves chicken LY6E as being required for initiation of MDV infection in a new host. Other laboratories have shown that LY6E binds to US10 and has been linked to MD resistance. US10 is a conserved herpesvirus virion protein as part of the tegument; therefore, we hypothesize US10 interaction with LY6E is required for LY6E-mediated endosomal escape of the MD nucleocapsid. Here, we will directly address the functional significance of the US10-LY6E interaction during horizontal transmission and whether this interaction is related to genetic MD resistance. To do this, we will first clone the LY6E gene from chicken lines determined to be relatively resistant, moderately resistant/susceptible, and highly susceptible to MD on the Illinois Poultry Farm using standard methods. The requirement of phosphorylation of US10 by UL13 on the US10-LY6E interaction will be determined using in vitro binding assays, as well as determining whether LY6E is incorporated as part of the MD virion during virus assembly in FFE cells, or whether US10 recruits LY6E following entry into a newly infected cell (host). Specific objectives include:Characterize the LY6E gene of different chicken lines.Determine that LY6E is part of the MD virion.Determine that LY6E and US10 interact during virus infection in cells.Determine that LY6E is important for horizontal transmission of MDV.