Viruses may acquire mutations that result in a tropism shift. RNA viruses, such as Coronaviruses (CoVs), are susceptible to tropism shifts. A tropism shift occurs when a virus alters the tissue or cell type it infects, which can have important implications for disease pathogenesis, virulence, transmission, and treatment control. Tropism shifts can occur after cross-species jumps, as well as result from within-host evolution. Beyond the human host, CoVs can be highly pathogenic to a wide variety of wildlife and companion animals. A spillover event from animals to humans, resulting in a tropism shift, has occurred at some point in the evolutionary history of all three highly pathogenic human CoVs: severe acute respiratory syndrome coronavirus (SARS-CoV), middle eastern respiratory syndrome coronavirus (MERS), and severe acute respiratory syndrome 2 (SARS-CoV-2). Therefore, studying the evolution of CoVs in non-human animals may be of critical importance for pandemic prevention. This was the focus of my dissertation, to apply state-of-the-art codon models of evolution to a variety of CoV viral sequences to identify how natural selection may alter viral proteins priming them for tropism shifts. Statistical codon models can infer both which codon sites and genes have been subject to positive or negative selection, effectively differentiating signal between random mutations and those that may impact fitness. These models may also compare selection at homologous sites between different phenotypes (i.e., Spike protein sequences isolated from the gastrointestinal tract and those from macrophages) to identify where selection is acting differently between the phenotypes. In chapter 2 I examined a CoV sequence isolated from hospitalized humans in Malaysia that resembled a Canine Coronavirus (CCoV) to investigate how natural selection had shaped the Spike protein sequence in related animal CoV sequences priming it to jump into humans. In chapter 3 I compared the natural selection signals at specific codon positions in the Spike protein from sequences isolated from two separate feline tropisms (gastrointestinal and macrophage) to identity which adaptive mutations may be associated with the tropism shift and subsequent shift in virulence. This was performed on Feline Coronavirus (FCoVs), where almost 90% of all wild and domestic cats are gastrointestinally infected with FCoVs, and infection becomes highly pathogenic as a result of the shift in tropism to the macrophages. Since intra-host evolution can impact tropism shifts, in Chapter 4 I performed a detailed high-throughout analysis of intra-host evolution of RNAseq data of Equine Coronavirus (ECoV), as well as natural selection analyses of related embecoviruses that have colonized the human host. Taken together, I report on novel signals of natural selection across viral proteins, with an emphasis on Spike, on a diverse set of CoV clades that shed light on the complexities of coronavirus evolution as it relates to tropism shifts. / Biology