A proof-of-concept study on the genomic evolution of Sars-Cov-2 in molnupiravir-treated, paxlovid-treated and drug-naïve patients

Abstract

Little is known about SARS-CoV-2 evolution under Molnupiravir and Paxlovid, the only antivirals approved for COVID-19 treatment. By investigating SARS-CoV-2 variability in 8 Molnupiravir-treated, 7 Paxlovid-treated and 5 drug-naïve individuals at 4 time-points (Days 0-2-5-7), a higher genetic distance is found under Molnupiravir pressure compared to Paxlovid and no-drug pressure (nucleotide-substitutions/site mean±Standard error: 18.7 × 10^-4 ± 2.1 × 10^-4 vs. 3.3 × 10^-4 ± 0.8 × 10^-4 vs. 3.1 × 10^-4 ± 0.8 × 10^-4, P = 0.0003), peaking between Day 2 and 5. Molnupiravir drives the emergence of more G-A and C-T transitions than other mutations (P = 0.031). SARS-CoV-2 selective evolution under Molnupiravir pressure does not differ from that under Paxlovid or no-drug pressure, except for orf8 (dN > dS, P = 0.001); few amino acid mutations are enriched at specific sites. No RNA-dependent RNA polymerase (RdRp) or main proteases (Mpro) mutations conferring resistance to Molnupiravir or Paxlovid are found. This proof-of-concept study defines the SARS-CoV-2 within-host evolution during antiviral treatment, confirming higher in vivo variability induced by Molnupiravir compared to Paxlovid and drug-naive, albeit not resulting in apparent mutation selection.

Fig. 1. SARS-CoV-2 RNA across time points in Molnupiravir-treated, Paxlovid-treated and Drug Naïve patients.

Each patient is depicted by lines of different colors. Day 2 is missing for ID7, ID17, and ID21. Day 5 is missing for ID20. Day 7 is missing for ID12 and ID13. SARS-CoV-2 RNA decay from Day 0 to Day 2, from Day 2 to Day 5 and from Day 5 to Day 7 is expressed as log median (IQR).

Fig. 2. Estimated maximum likelihood phylogenetic tree of the SARS-CoV-2 strains from Molnupiravir-treated, Paxlovid-treated and drug naïve individuals.

The phylogeny was estimated with IqTree2 using the best-fit model of nucleotide substitution TN + F + G4 with 1000 replicates fast bootstrapping. Leaves number represents the sample ID, bootstrap values higher than 90 are shown on branches, color of branches represents the different patients whose ID is also reported next to the tree with brackets. Information regarding treatment, lineage, SARS-CoV-2 viral load and the time points analyzed were reported in the annotation columns in the right-hand side of the figure. Tree scale is expressed as nucleotide substitutions per site.

Fig. 3. Circos plots representing the SNPs found in SARS-CoV-2 strains.

Panels represent SARS-CoV-2 strains found in (a) Molnupiravir-treated, (b) Paxlovid-treated and (c) drug-naïve individuals across time points. Each time point is defined by one track and grouped according to patient. Tracks in black represent timepoints not available; striped tracks represent samples not sequenced as viral load was undetectable. SNPs are defined by different colors, shapes and size according to type of nucleotide substitution (red: G-A; orange: C-T; green: other), non-synonymus (circle) or synonymus mutations (triangle), and intra-patient prevalence, respectively. The outside track corresponds to SARS-CoV-2 genome architecture. Overlapping orfs are not reported and comprise: orf2b (coordinates 21744–21860) overlapping S; orf3b (coordinates 25814–25879), orf3c (coordinates 25457–25579), orf3d (coordinates 25524–25694) and orf3d-2 (coordinates 25596–25694) overlapping orf3a; orf9b (coordinates 28284–28574) and orf9c (coordinates 28734–28952) overlapping N.

Fig. 4. RdRp amino acid mutations that emerged during Molnupiravir treatment across time points.

Representation of the structure of the SARS-CoV-2 RdRp with Molnupiravir/NHC complex (PDB:7OZV). Amino acid positions corresponding to the Molnupiravir-induced mutations are shown as spheres of different colors based on day of appearance and relative abundance: (i) mutations appearing at day 2 with relative abundance between 2 and 10% in light green, and between 10 and 20% in green; (ii) mutations appearing at day 5 with relative abundance between 2 and 10% in light blue, and between 10 and 20% in blue; (iii) mutations appearing at day 7 with relative abundance between 2 and 10% in light red, between 10 and 20% in red, and between 20 and 40% in dark red; (iv) mutations appearing at both day 2 and day 5 in cyan (including the G13604A-R55H mutation); (v) mutations appearing at day 2 or day 7 in yellow; vi) mutations appearing at day 5 or day 7 in purple. Template RNA is colored in black, while product RNA in olive. RdRp: RNA-dependent RNA polymerase.