Genotyping amplicons from SARS-CoV-2 viruses to verify the identity of PCR products

We used endpoint PCR to verify the presence of a new subgenomic RNA in SARS-CoV-2 (termed N.iORF3) and verify using nanopore sequencing that this is expressed via a newly evolved transcription regulatory sequence (TRS). We further show that this encodes 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.