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 ORFs, thereby deleting endogenous gene functions. An alternative strategy is to manipulate viral RNA synthesis by inserting copies of the viral transcription regulatory sequence (TRS), which drives the transcription of viral subgenomic RNAs. However, coronavirus transcription is tightly regulated, and these modifications frequently disrupt native subgenomic RNA synthesis and attenuate viral growth. Here, we describe 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 minimize off-target effects to transcription. This virus is genetically stable, with WT growth kinetics and unaltered subgenomic RNA transcriptional ratios. We developed a flexible reverse genetics system, which allows rapid cloning and virus recovery, supported by optimized HCoV-OC43 culture conditions for high-titre stock generation, 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 based on active viral translation. We found that transcriptional responses to infection of A549 human lung epithelial cells were predominantly inflammatory, rather than interferon-mediated, and that bystander cells upregulated pathways associated with cytokine response signalling and cell-cell contact sensing. Together, these tools expand the experimental utility of HCoV-OC43, an important seasonal respiratory pathogen and low-containment model for betacoronavirus biology.