Establishment of a human coronavirus HKU1 infection evaluation system using apical-out airway organoids

Human coronavirus HKU1 (HCoV-HKU1) is difficult to study because it does not propagate in standard cell lines. Consequently, research has relied on the air-liquid interface model, which is limited by long culture periods and the absence of cytopathic effects (CPEs). To address these challenges, we established an infection evaluation system using apical-out airway organoids (AOAOs). Derived from human bronchial epithelial cells, AOAOs supported efficient HCoV-HKU1 replication, and immunofluorescence revealed a preferential infection of ciliated cells. Notably, HCoV-HKU1 induced a distinct CPE characterized by organoid dissociation, enabling the quantification of viral titres via the TCID₅₀ assay. Applying this model to antiviral testing, the serine protease inhibitor nafamostat effectively inhibited viral infection and CPE, consistent with the known entry mechanism. Thus, AOAOs provide a rapid, scalable and reproducible platform for HCoV-HKU1 research, overcoming limitations of traditional systems. This approach enables reliable viral replication analysis, titre determination and drug evaluation and may accelerate research on difficult-to-culture respiratory viruses.