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Background Molnupiravir is an orally available prodrug of the antiviral nucleoside analog N-Hydroxycytidine (NHC). In preclinical studies NHC has shown broad-spectrum antiviral activity against multiple RNA viruses including SARS-CoV-2. Incorporation of NHC by viral polymerases impairs replication by introducing errors into the viral genome. NHC has been shown to have a high barrier to the development of resistance in vitro with RSV, Influenza and Venezualen Equine Encephalitis viruses. In these studies, we have explored the potential for SARS-CoV-2 to develop resistance to NHC in cell culture. Methods Vero E6 cells were infected with SARS-CoV-2 (WA-1) in triplicate in the presence of NHC or a C3L-protease inhibitor (MRK-A). Culture supernatants from wells with the highest drug concentration exhibiting a cytopathic effect (CPE) score of ≥ 2+ were repassaged and at each passage, IC50 values were estimated based on CPE scoring. At each passage, full genome next generation sequencing (NGS) was performed on the viral RNA Results No change in susceptibility to NHC (EC50 fold change ≤ 1.1) was noted in 2 of 3 cultures and a 2-fold change was observed in one culture after 30 passages. In contrast, a 3- to 4-fold decreases in susceptibility to the 3CL protease inhibitor were seen by passage by 12, with increasing resistance of 4.6- to 15.7-fold observed by passage 30. NHC passaged viruses exhibited 53 to 99 amino acid changes, including substitutions and deletions (both in-frame and frameshift), across 25 different viral proteins as compared with 10 to 13 changes in 13 proteins in the MRK-A cultures. With NHC, 3 to 4 changes were observed in the viral polymerase; however, these were randomly distributed, and none were observed more than once. In contrast, the 3CL protease passaged virus had a nsp5 T21I substitution detected in all 3 cultures. Conclusion No evidence of SARS-CoV-2 phenotypic or genotypic resistance was observed following 30 passages with NHC. A random pattern of amino acid changes were observed across multiple proteins consistent with the mechanism of action of NHC. In the same study, resistance was readily selected to a control 3CL protease inhibitor. Together these data support previous reports demonstrating the high barrier to resistance of NHC. Disclosures Julie Strizki, PhD, Merck and Co.: Stocks/Bonds Nicholas Murgolo, PhD, MERCK: Employee|MERCK: Stocks/Bonds John Howe, PhD, Merck & Co Inc: Employee|Merck & Co Inc: Stocks/Bonds Beth Hutchins, PhD, Merck & Co.: Employee|Merck & Co.: Stocks/Bonds Hiroshi Mohri, PhD/MD, Merck: Grant/Research Support Daria Hazuda, PhD, Merck: Employee|Merck: Stocks/Bonds Jay Grobler, PhD, Merck & Co., Inc.: Employee|Merck & Co., Inc.: Stocks/Bonds. |