Alphaviruses are positive-sense single-stranded RNA viruses primarily transmitted by mosquito vectors and are responsible for numerous human and veterinary diseases. Traditionally, investigation of viral determinants that drive alphavirus transmission and pathogenesis has focused on protein-coding changes in viral proteins. However, the role of viral RNA secondary structure in viral emergence remains poorly understood. We have developed a computational method to compare RNA structures across the alphavirus genus to identify RNA structure signatures associated with specific viral properties and subtypes (e.g. pathogenic vs attenuated strains). Using this approach, we have identified several regions in the genomes of Venezuelan equine encephalitis virus and Sindbis virus that are predicted to contain RNA structures important for viral emergence and pathogenesis. Using molecular and biochemical approaches, we have defined functional roles for several of these RNA structures in translation of viral RNA as well as cellular tropism. Interestingly, despite encoding distinct RNA structures, pathogenic/epizootic strains of diverse alphavirus species replicate differentially in myeloid cells, suggesting that myeloid cell replication fitness may be a hallmark of alphavirus emergence. Changes in myeloid cell replication fitness were also linked to alterations in pathogenesis in a small animal model. Using proteomics, we have identified several RNA-binding proteins that differentially interact with these viral RNA structures, indicating that distinct molecular mechanisms contribute to altered myeloid cell replication. Currently, we are investigating how altered myeloid cell replication contributes to transmission and pathogenesis in other alphavirus species, and how this knowledge may be leveraged for enhanced surveillance and development of RNA therapeutics.