Table 4.

Remdesivir EC₅₀ Phenotyping Against Clinical Isolates With Emergent Nsp12 Amino Acid Substitutions and SDMs

Clinical Isolates	Remdesivir EC50 (μM)			EC50 Fold Change (SD) From Reference
Substitution	First Replicate	Second Replicate	Mean (SD)
Clinical isolates (frequency of substitution, %)
Wild-type SARS-CoV-2 (WA1)a	0.26	0.21	0.24 (0.04)	1.0
A16V (88.6%)	0.19	0.16	0.18 (0.02)	0.8 (0.05)
C799F (22.4%)	0.60	0.57	0.58 (0.03)	2.5 (0.30)
V792I (99%)b	0.50	0.50	0.50 (0.0)	2.2 (0.36)
S759A + V792I positive controlc	2.82	2.49	2.66 (0.23)	11.4 (0.90)
SDMs
Wild type, SH01d	15.37	13.87	14.62 (1.06)	1.0
K59N	14.7	14.04	14.37 (0.47)	1.0 (0.04)
K59N + V792I	49.82	49.61	49.72 (0.15)	3.4 (0.24)
V792I	45.9	46.01	45.96 (0.08)	3.2 (0.23)
D684N	No replication	No replication	NA	NA
V764L	No replication	No replication	NA	NA

Abbreviations: EC₅₀, half maximal effective concentration; NA, not applicable; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SD, standard deviation; SDM, site-directed mutant.

^aWA1: clinical isolate with wild-type reference SARS-CoV-2 lineage A strain from January 2020 in Washington State.

^bK59N and V792I substitutions in Nsp12 emerged at day 10 in participant COV.01517. P323L in Nsp12 was observed at baseline and at all time points sequenced including day 10. Culturing of the virus was attempted twice and, in both cultures, K59N reverted to wild type. Phenotyping was conducted on the cultured virus containing P323L and V792I.

^cThe positive control used for these experiments contained the Nsp12 S759A and V792I substitutions and was generated using recombinant SARS-CoV-2 virus. These substitutions were previously observed in an in vitro resistance selection experiment using GS-441524 (the parent nucleoside of remdesivir [21]).

^dSH01: wild-type reference SARS-CoV-2 replicon generated from clinical isolate from Shanghai (lineage B).