High Throughput Genetics And Characterization Of An RNA Arbovirus, Sindbis Virus, Using Accurate Next-Generation Sequencing Of Viral Evolution And RNA Enrichment

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The goal of these studies was to investigate Sindbis virus adaptation to various infection bottlenecks and utilize the dynamics of minor variants to study viral genetics in a high-throughput manner. During infection an RNA virus exists as a tremendously diverse population, and this genetic diversity underlies their ability to rapidly adapt to new conditions and cause disease. Since viruses evolve as populations, our understanding of viral evolution has historically been limited by the inability to characterize populations. Whilst new sequencing technologies provide sufficient depth to sequence full viral populations, their intrinsic base-calling error rate combined with mutations introduced during sample processing makes viral mutations and sequencing errors indistinguishable. Utilizing rolling reverse transcription, the novel CirSeq technique virtually eliminates sequencing errors by bioinformatically parsing tandem generated repeats, and for the first time allows a highly accurate mutational landscape profile of the whole viral population. In addition, a novel hybridization capture technique we developed allows us to maximize the sequencing coverage of desired viral RNA molecules. We used these technologies to map the mutational distribution of RNA virus populations and perform genetics are previously unseen scales. We sequenced serial passages of the well-characterized Sindbis virus to yield novel information on genetic features crucial for viral replication. We analyzed how the starting in vitro transcribed RNA population adapts to various bottlenecks encountered during electroporation and subsequent passaging, and during packaging and egress. Then we compared these data to previous studies of critical genome sites and expanded our study to new sites of interest. We posit that such unbiased high-throughput genetics pushes the envelope beyond the previous limits on discovery of viral functional elements. These techniques can be used to further characterize clinically relevant RNA viruses that are agents of current and recent epidemics, such as SARS-CoV-2 coronavirus, chikungunya virus, Zika virus, eastern equine encephalitis virus, dengue virus, and West Nile virus

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alphavirus; arbovirus; coronavirus; RNA; sindbis


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Government Document




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Attribution-NonCommercial-NoDerivatives 4.0 International


dissertation or thesis

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