Investigating the Pathogenesis of HCoV-NL63 in Bronchial/Tracheal and Small Airway Human Epithelial Cells: Monitoring of Cell Viability, Cell Cycle, Virus Replication, and Cytokines Production

• Small airway epithelial cells are more susceptible to HCoV-NL63 infection than bronchial/tracheal cells. • There are significant differences in cell viability, viral replication dynamics and cytokine production between the two cell types, with SA-HEC exhibiting greater susceptibility and cytokine production compared to BT-HEC. • In SA-HEC cell lines, HCoV-NL63 peak titer happened latter than what is observed in LLC-MK2 cells, day 16 vs day 5-6. As seen in children infected with HCoV-NL63, with observed virus shedding lasting up to three weeks. • ALI SA-HEC is a better model of infection for HCoV-NL63. • These findings contribute to our understanding of the pathogenesis of HCoV-NL63 infection and suggest potential therapeutic targets for combating severe coronavirus infections. Human airway tissue models serve as invaluable tools for investigating the infection of respiratory viruses. In this study, we focused on the interaction between Human coronavirus (HCoV)-NL63, a common cold causative agent, and primary bronchial/tracheal (BT) and small airway (SA) human epithelial cell (HECs). The HCoV-NL63 virus, which uses angiotensin-converting enzyme 2 (ACE2) as a receptor by binding the viral spike (S) protein and facilitating cell entry. We inoculated human BT-HEC and SA-HEC with 1 × 10 5 pfu of HCoV-NL63, and monitored cell viability, cell cycle, viral replication, and cytokine production over a 16-day experiment. Throughout the study, BT-HEC culture maintained approximately 80% cell viability, while SA-HEC culture exhibited a decline from 88% to 79% cell viability compared to the control and inoculated groups. Also, HCoV-NL63 infection induced more pronounced cell cycle disruption in SA-HEC, with virus-induced G2/M progression reaching 9.37% compared to modest increases in BT-HEC. In BT-HEC, virus replication gradually decreased, becoming undetectable after Day 10. Conversely, in SA-HECs, a statistically significant increase in virus replication was observed after Day 13. Furthermore, in BT-HEC, the levels of IP-10 and IL-6 showed significant upregulation compared to the control group. Similarly, in SA-HEC, IP-10, IL-6, and MIP1-α exhibited significant upregulation compared to the control group. This study underscores the significance of airway HEC culture models, which closely resemble human airway architecture and physiology. Such models contribute to a better understanding of viral replication, pathogenesis, and host immune responses in the context of the infection site. Additionally, they provide a foundation for developing therapeutic interventions against current and future emerging coronaviruses.