Genotyping amplicons from SARS-CoV-2 viruses with or without mutations to the N.iORF3 transcription regulatory sequence to determine relative viral fitness in A549-ACE2-TMPRSS2 cells

We have demonstrated that a newly evolved TRS within the Nucleocapsid gene of SARS-CoV-2 (termed N.iORF3) leads to the expression of a novel subgenomic mRNA encoding a truncated C-terminal portion of Nucleocapsid, which is an antagonist of type I interferon production. Using reverse genetics-derived viruses we show N.iORF3 contributes to viral fitness during infection and observe distinct phenotypes when the Nucleocapsid coding sequence is mutated compared to when the TRS alone is ablated. Competition assays were performed to determine the relative fitness of different virus mutants and amplicons were analysed to quantify the proportions of different viruses in tissue culture.