Fig. 3

Recombinant SARS-CoV-2 S:E484 mutants show distinct replicative fitness in competition assays against wild type in the absence of neutralizing antibodies. Calu-3 cells were inoculated in triplicate with a 10,000 PFU containing mixture of two competing E484 variants or WT at two or three defined ratios (all or two of ratios 1:9, 1:1, 9:1). At 24 hpi, 100 µl of virus containing supernatant were transferred to naïve Calu-3 cultures for a total of five (A-C) or three (D-I) consecutive passages. Viral RNA was isolated from the initial inoculum (p0) and from culture supernatants at passage 1 (p1), 3 (p3), and 5 (p5) and subjected to one-step reverse transcription to generate an 800-bp PCR fragment for Sanger-sequencing. The three columns of plots show the p0 virus inoculum ratios of 1:9 (left), 1:1 (middle), and 9:1 (right) (p0 means a back-titration of the inoculum applied to the cells). Colors indicate virus mixture and identity, according to the legend on the right of each row (A-I). Peak heights in sequencing chromatograms were analyzed using the ChromatQuantitator online tool. WT = wildtype.