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.