Reoviridae viruses, which include important human and animal pathogens, assort and package nine to twelve positive-sense RNA segments that are converted to genomic double-stranded RNA during virus assembly in cytoplasmic inclusions. Reoviridae genetic diversity contributes to host range and vaccine responses. To infect and adapt, Reoviridae viruses have evolved mechanisms to promote genetic diversity and complement defective particles. These mechanisms include segment reassortment and transmission of multiple particles as collective infectious units. Evolutionary benefits of diversity are countered by a need to maintain interactions mediating multipartite genome packaging, assembly, egress, and transmission. Thus, the replication strategy also may inherently constrain diversity. The goal of the proposed research is to understand how Reoviridae assembly and transmission processes regulate unique aspects of viral genetic diversity. To accomplish this goal, we will use reovirus, a genetically tractable Reoviridae virus with established tissue culture and mouse models. The termini of Reoviridae RNA segments are important for packaging, but RNA elements that mediate assortment of a specific collection of segments are poorly defined. In Specific Aim 1, we will sequence defective viral gene segments using long-read and short-read approaches to identify minimal reovirus RNA packaging and assortment determinants. We will determine the capacity of RNA recognition elements to interchangeably mediate segment packaging and assortment using reverse genetics and functional assays. Physical sequestration of viral RNA in cytoplasmic inclusions may influence segment reassortment during coinfection, and innate cellular responses may influence reassortment by inhibiting superinfection. In Specific Aim 2, we will determine the localization of viral RNA during coinfection using sensitive RNA imaging probes and effects of infection timing on replication and reassortment in vitro and in vivo using viruses encoding silent genetic polymorphisms. Transmission of Reoviridae viruses in extracellular vesicles may promote simultaneous multi-particle infection of target cells. In Specific Aim 3, we will elucidate contributions of vesicle- mediated virus transmission to genetic diversity and virulence. The proposed studies will provide insight into mechanisms of viral genetic diversity that are mediated by the assembly and transmission processes of viruses in the Reoviridae family. Many principles derived from this work will apply broadly to viruses that replicate in compartmentalized subcellular regions, induce innate antiviral responses, or travel in extracellular vesicles. Together, these findings will promote rational engineering of Reoviridae-based preventives and therapeutics and identification of factors that predict outcomes of natural virus coinfection and transmission.