Extended Data Fig. 2: Genetic stability of YF-S0 during passaging in BHK-21 cells.

a, Schematic of YF-S0 passaging in BHK-21 cells. YF-S0 vaccine virus recovered from transfected BHK-21 cells (P0) was plaque-purified once (P1) (n = 5 plaque isolates), amplified (P2) and serially passaged on BHK-21 cells (P3–P6). In parallel, each amplified plaque isolate (P2) (n = 5) from the first plaque purification was subjected to a second round of plaque purification (P3*) (n = 25 plaque isolates) and amplification (P4*). b, Schematic of tiled RT–PCR amplicons from three different primer pairs used for detection of the inserted SARS-CoV-2 S viral RNA sequence present in supernatants of different passages. All data are from a single representative experiment. c, RT–PCR fingerprinting performed on the virus supernatant collected from serial passage 3 (P3) and 6 (P6) of plaque-purified YF-S0. d, Immunoblot analysis of S expression by P3 and P6 of YF-S0. e, RT–PCR fingerprinting on amplified plaque isolates from the second round of plaque purification (P4*), 20 individual amplified plaque isolates are shown here (1–20). c, e, Control, YF-S0 cDNA (0.5 ng); ladder, 1-kb DNA ladder. Direct Sanger sequencing confirmed maintenance of full-length S inserts for 25 out of 25 plaques (100%). After two rounds of plaque purification and amplification, only in three isolates a single point mutation was found (two silent mutations and one missense mutation resulting in a S47P amino acid change in the N terminus of S1); at a low <10⁻⁴ mutation frequency (that is, 3 nt changes observed in a total of 25 × 4,196 nt = 104,900 nt sequenced; of which 25 × 3,780 nt = 94,500 nt were of S transgene sequence). This mutation rate is similar to that of parental YF17D under current vaccine manufacturing conditions^14,78. For gel source data, see Supplementary Fig. 1.