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Remdesivir (RDV) is used widely for COVID-19 patients despite varying results in recent clinical trials. Here, we show how serially passaging SARS-CoV-2 in vitro in the presence of RDV selected for drug-resistant viral populations. We determined that the E802D mutation in the RNA-dependent RNA polymerase was sufficient to confer decreased RDV sensitivity without affecting viral fitness. Analysis of more than 200,000 sequences of globally circulating SARS-CoV-2 variants show no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we also observed changes in the Spike (i.e., H69 E484, N501, H655) corresponding to mutations identified in emerging SARS-CoV-2 variants indicating that they can arise in vitro in the absence of immune selection. This study illustrates SARS-CoV-2 genome plasticity and offers new perspectives on surveillance of viral variants. One Sentence SummarySARS-CoV-2 drug resistance & genome plasticity

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