Long-term Effects of SARS-CoV-2 on Ciliary Function and Ciliogenesis

Introduction: Cilia are critically important in the mucociliary clearance of the airways. This study explored long-term effects of SARS-CoV-2 on ciliary regeneration and function. Aim: We aimed to investigate respiratory epithelial recovery and ciliary function in individuals 3-12 months post SARS-CoV-2 infection. Methods: Three post-COVID cohorts; the first of which (FOLLOW n=41) underwent nasal epithelial cell sampling 3-12 months after the first waves of SARS-CoV-2 infection in both hospitalised and community settings, and self reported Long-COVID symptoms were recorded. The second cohort, (ULTRON n=15) contained patients 3-12 months post-Omicron variant infection, all of whom were vaccinated; the third cohort (PROSAIC n=46) had RNA extracted from nasal brushings 6-12 months after recovery from severe infection with the pre-omicron variant of SARS-CoV-2. In the first two cohorts, ciliary function was assessed using high-speed-video-microscopy, with ultrastructural analysis assessed by Transmission Electron Microscopy. Expression of the ciliogenesis gene, FOXJ1, was measured by qRT-PCR in the third cohort. Additionally data of other virus and bacterial positive nasal brushings were analysed to ascertain the effect of a multitude of other infections on cilia function to contextualise post-COVID’s effect on cilia. Results: There was significant ciliary dysfunction within early pandemic SARS-CoV-2 patients, persisting up to a year post-infection. 90% of individuals had ultrastructural defects, marked by mislocalised basal bodies and intracytoplasmic cilia up to 12 months post infection; without correlations with any long-term COVID-19 symptoms. With post-Omicron infected, vaccinated cohort, ciliogenesis was normal, and no mislocalised basal bodies nor intracytoplasmic cilia were seen. Across cohorts, defects in cilia function were present compared to pre pandemic controls: Reduced cilia beat frequency (FOLLOW p<0.01), reduced amplitude per second: (FOLLOW p<0.01, ULTRON p<0.01). Foxj1 expression post-COVID was significantly reduced 6 months (p<0.001) and 12 months (p=0.002) within pre-omicron-variant infection, compared with 8 healthy volunteers, with some recovery of Foxj1 mRNA at 12-months. A clinical cohort investigated for primary ciliary dyskinesia and found to be PCD Negative showed no ciliary defects from other viral/bacterial infections. Conclusion: Persistent cilia loss, and ciliogenesis defects post COVID are explained by a reduction in FOXJ1 gene expression, affecting ciliogenesis and ciliary function. There are differences observed between early and Post Omicron infections, with vaccinated individuals being less affected, although some cilia impairments persist. There is a long-term cilia movement restriction following SARS-CoV-2, and specific to infections 2020-2021, is a decreased cilia beat frequency. Exploring the virus’s long-term effects on gene expression is merited, and understanding the viral kinetics of FOXJ1 expression may shed light on how this is impaired, and provide insights into Long-COVID clinical manifestations.