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

Insertion of fluorescent reporter genes into viral genomes is a powerful tool for monitoring infection. 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. This virus is genetically stable, with wildtype growth kinetics and unaltered subgenomic RNA transcriptional ratios. We developed a flexible reverse genetics system, which allows rapid cloning and virus recover, supported by optimised HCoV-OC43 culture conditions, which support high titre stock growth, and validated analytical reagents. Our reporter virus enabled sensitive detection and isolation of infected cells, facilitating transcriptomic analyses that distinguish host responses in infected and bystander populations. Together, these tools expand the experimental utility of HCoV-OC43, an important seasonal respiratory pathogen and low containment model for betacoronavirus biology.