Molecular Surveillance of Coronaviruses in Riyadh (2025-2026): Persistent Genotype C and Conserved N-Glycosylation...

Seasonal human coronaviruses (HCoVs) continue to undergo adaptive evolution under structural and immune-mediated constraints. We investigated the molecular epidemiology and spike (S) protein structural variation of circulating coronaviruses in Riyadh, Saudi Arabia, during the 2025-2026 winter season, with particular emphasis on genotype persistence and glycosylation architecture in HCoV-OC43. Among 293 nasopharyngeal aspirates (NPAs) collected from hospitalized patients with acute respiratory illness, HCoV-OC43 was detected in 26 cases (8.87%), whereas other seasonal coronaviruses were not identified. Partial sequencing of the S gene revealed 97.84-98.23% nucleotide identity relative to the prototype strain VR-759, with amino acid substitutions distributed at discrete positions rather than within extended variable domains, indicating structural conservation. Phylogenetic reconstruction demonstrated that all Riyadh isolates clustered within genotype C, together with previously circulating local strains, supporting sustained endemic persistence and in situ evolution. In silico analysis of the S protein glycosylation landscape identified four invariant N-linked glycosylation motifs (N-X-S/T) at residues 46, 121, 134, and 190, reflecting strong structural constraints on glycan-dependent folding and antigenic configuration. A genotype-associated K68N substitution generated an additional N-glycosylation motif (68NGTD) in multiple Riyadh isolates, potentially modifying local glycan shielding without disrupting the overall glycosylation framework. The preservation of core glycosylation sites alongside selective motif acquisition suggests evolutionary fine-tuning of S surface topology rather than large-scale structural remodeling. Collectively, these findings indicate that genotype C persistence in Riyadh is accompanied by conserved S architecture and subtle glycosylation adjustments that may modulate immune recognition while maintaining structural integrity. Continued high-resolution molecular surveillance will be critical for defining the functional consequences of S microevolution in endemic HCoVs.