Kyla Tozer, University of Toronto

Kyla Tozer1,2, Maedah Naghibosadat1, Henry Wong2,3, Jad Tirani3, Kayla Gaete1, Calvin Sjaarda2,3, Danielle Brabant-Kirwan4, Ramzi Fattouh5, Prameet M. Sheth2,3, Robert Kozak1 Affiliations: 1. Sunnybrook Health Sciences Centre, Biological Sciences Platform, Toronto, Ontario 2. Queen's University, Kingston, Ontario 3. Kingston Health Sciences Center, Kingston, Ontario 4. Health Sciences North, Sudbury, Ontario 5. Unity Health, Toronto, Ontario

Discovering & Evolving

Introduction: Human coronavirus OC43 (HCoV-OC43) causes seasonal respiratory infections and enters host cells through spike-mediated binding to 9-O-acetylated sialic acids. Antigenic drift within the spike receptor-binding domain (RBD) may limit cross-reactive immunity; however, the genetic epidemiology of HCoV-OC43 and drivers of RBD variation remain poorly defined. We sequenced clinical HCoV-OC43 isolates to characterize seasonal RBD variability and performed in vitro passaging to examine genomic changes in the absence of immune pressure to investigate viral adaptation and inform seasonal coronavirus vaccine design. Methods: Clinical HCoV-OC43 samples across Ontario (2021-2024) were whole-genome sequenced (WGS) on the GridION platform and analyzed using a custom bioinformatics pipeline with NCBI spike annotation. To assess immune-independent evolution, HCoV-OC43 was serially passaged 13 times in HCT-8 cells, and each passage underwent WGS. Results: A total of 366 HCoV-OC43-positive clinical samples were collected from three ambulatory care centers (172 female, 77 male, 57 NA; ages 7 months–100 years). Ct values ranged from 12.37 to 34.73 (median 24.63). Genomic variability was highest within spike positions 1200–1800, with 65.4% of mutations localized to the S1 region and 46.2% to the N-terminal domain. Serial passage of HCoV-OC43 identified the emergence of a single receptor-binding domain mutation substitution at Q487R after passage 9. Conclusion: We identify seasonal antigenic drift in the HCoV-OC43 spike protein that may limit cross-reactive immunity. In vitro passaging with WGS shows receptor-binding domain variation can arise independently of immune pressure, highlighting intrinsic viral adaptation and supporting genomic surveillance–informed vaccine strategies for seasonal coronaviruses.