In Vitro Selection Of Remdesivir Resistance Suggests Evolutionary Predictability Of Sars-Cov-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in Spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.

Author summaryThe emergence of SARS-CoV-2 has led to a worldwide pandemic with significant morbidity and mortality. Remdesivir is the only antiviral with FDA approval for treatment. Antivirals use comes at a risk, as viruses may acquire mutations overcome the inhibition. We identified a mutation in the virus polymerase responsible for decreased sensitivity to Remdesivir. A change at this conserved site was not predicted, and the mutation did not cause a replication advantage or change in sensitivity to another antiviral drug. Importantly, this change occurred at very low frequency globally. Unexpectedly, passage of SARS-CoV-2 led to an accumulation of mutations in Spike. A number occurred at the same sites but to different residues as those in emerging variants of concern indicating they arise in the absence of immune pressure. Our data indicate low-level Remdesivir resistance in SARS-CoV-2 is different to other RNA viruses and monitoring changes in vitro provides insight into general virus adaptation of newly emerging viruses.