A stable subgenomic reporter coronavirus enables transcriptional profiling of bystander cells.

In coronaviruses, this is commonly achieved by replacing accessory open reading frames, thereby deleting endogenous gene functions. An alternative strategy is to manipulate viral transcription by inserting copies of the viral transcription regulatory sequence (TRS) which drive viral subgenomic RNA transcription.

However, coronavirus transcription is tightly regulated, and these modifications frequently disrupt native subgenomic RNA synthesis and attenuate viral growth. Here, we report a reporter coronavirus that overcomes these limitations. Using human coronavirus (HCoV)-OC43 as a model system, we inserted an mNeonGreen reporter between the Spike and ORF5 coding regions, engineering the TRS and surrounding sequence to minimise off-target effects to transcription.