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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\u2013cell contact sensing. 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of General Virology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.microbiologyresearch.org\/content\/journal\/jgv\/10.1099\/jgv.0.002282?crawler=true&mimetype=application\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,18]],"date-time":"2026-06-18T15:17:27Z","timestamp":1781795847000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.microbiologyresearch.org\/content\/journal\/jgv\/10.1099\/jgv.0.002282"}},"subtitle":["&lt;xhtml:span xmlns:xhtml=\"http:\/\/www.w3.org\/1999\/xhtml\" xml:lang=\"en\"&gt;&lt;p&gt;This article is part of our &lt;span style=\"color: rgb(33, 51, 104);\"&gt;&lt;a href=\"\/content\/emerging-investigators-collection-2025-26\"&gt;Emerging Investigators collection&lt;\/a&gt;&lt;\/span&gt;.&lt;\/p&gt;&lt;p style=\"line-height: 1.2;\"&gt;&lt;img src=\"\/upload\/emerging\/Harriet_Mears.png\" alt=\"photograph of Harriet Mears\" class=\"ImageBorder img-responsive eb-people fr-fic fr-dib\" style=\"width: 120px; float: left; margin: 0 10px 10px 0;\" \/&gt;&lt;span style=\"font-size: 12px; font-weight: normal;\"&gt;&lt;strong&gt;Harriet Mears&amp;#160;&lt;\/strong&gt;completed her PhD at the University of Cambridge, where she examined the biochemical interactions which underly innate immune antagonism of viral translation. She moved to the Francis Crick Institute as a postdoctoral fellow, where she investigated novel gene expression in SARS-CoV-2, optimised culture systems for seasonal coronavirus research, and began to explore how coronavirus package their genomes into new virus particles. She is currently a Lecturer in Virology at the University of Bristol, where her research examines how RNA structure and RNA-protein interactions regulate viral lifecycles. She is using biochemical and sequencing approaches, coupled with viral reverse genetics, to understand how positive-sense RNA viruses regulate genome packaging and particle assembly.&lt;\/span&gt;&lt;\/p&gt;\n&lt;\/xhtml:span&gt;"],"short-title":[],"issued":{"date-parts":[[2026,6,18]]},"references-count":120,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2026,6,18]]}},"URL":"https:\/\/doi.org\/10.1099\/jgv.0.002282","relation":{},"ISSN":["0022-1317","1465-2099"],"issn-type":[{"value":"0022-1317","type":"print"},{"value":"1465-2099","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,18]]}}}