Molnupiravir, an oral prodrug of N-hydroxycytidine (NHC), previously demonstrated broad in vitro antiviral activity against multiple RNA viruses and has shown a high barrier to the development of resistance. Here, we present the antiviral activity of NHC against recent SARS-CoV-2 variants and the results of resistance selection studies to better understand the potential for viral resistance to NHC. NHC activity against SARS-CoV-2 variants omicron (BA.1, BA.1.1, BA.2, BA.4, BA.4.6, BA.5, BQ.1.1, XBB.1, and XBB.1.5), alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), and mu (B.1.621) was evaluated in Vero E6 cells using cytopathic effect assays. Resistance selection studies were performed by passaging SARS-CoV-2 (WA1) in the presence of NHC or a 3C-like protease inhibitor (MRK-A) in Vero E6 cells. Supernatants from cultures exhibiting a cytopathic effect score of ≥2 were re-passaged, and IC50 values were estimated. Whole-genome deep sequencing was performed on viral RNA isolated at each passage. NHC demonstrated similar potency against all SARS-CoV-2 variants evaluated. No evidence of SARS-CoV-2 phenotypic or genotypic resistance to NHC was observed following 30 passages. A random pattern of nucleotide changes was observed in NHC cultures, consistent with the drug's mechanism of action. In contrast, resistance was readily selected in all three MRK-A control cultures with the selection of a T21I substitution in the 3C-like protease. In conclusion, molnupiravir maintains antiviral activity across all major SARS-CoV-2 variants. Furthermore, no evidence of viral resistance to NHC was observed, supporting previous reports that NHC has a high barrier to developing resistance.
Keywords: COVID-19; N-hydroxycytidine; antiviral resistance; molnupiravir.