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. 2022 Jul 27;8(2):veac063. doi: 10.1093/ve/veac063

Figure 1.

Figure 1.

In vitro evolution of SARS-CoV-2QLD02 to mACE2 utilization. (A) Schematic of SARS-CoV-2QLD02 passaging in HEK293T-hACE2 or HEK293T-mACE2N31K/H353K cells, co-cultured with HEK293T-mACE2 cells (nine passages), followed by passaging in HEK293T-mACE2 cells (three passages) and mouse infections. Viruses used to infect mice and viruses derived from mice lungs (2 dpi) were sequenced. Stock virus was prepared for MA1 and MA2 from mouse lungs (2 dpi) for use in subsequent experiments. (B) Supernatants from passages 4 and 9 (from the co-cultures—blue arrows) were used to infect HEK93T-mACE2 cells and viral growth over 72 h determined by CCID50 assays. Dotted line—limit of detection. MA1, MA3, and MA6 (underlined) were derived from HEK293T-hACE2/HEK293T-mACE2 co-cultures, MA2, MA4, and MA5 from HEK293T-mACE2N31K/H353K/HEK293T-mACE2 co-cultures. (C) Inverted light microscopy images of CPE in HEK293T-mACE2 cells infected with passage 9 supernatants at 72 h postinfection. Images are representative of at least three replicates. (D) MA viruses obtained after three passages in HEK293T-mACE2 cells were used to infect C57BL/6J mice. Lung tissue titers are shown for 2 dpi for MA1-5 (n = 1 for each) and for SARS-CoV-2QLD02 (n = 6).