Understanding the Mechanisms of Oseltamivir Resistance Evolution in H3N2 Influenza Strains Circulating in Nigeria Under Selective Pressure

Oseltamivir resistance in seasonal influenza viruses is a global antiviral surveillance priority, given the clinical reliance on neuraminidase inhibitors for influenza treatment, particularly in immunocompromised patients. The mechanisms and evolutionary trajectory of oseltamivir resistance development in H3N2 strains circulating in Nigeria, under conditions of real-world drug pressure in clinical settings, remain entirely uncharacterised. This study investigates the mechanisms and evolutionary dynamics of oseltamivir resistance in Nigerian H3N2 isolates using a combined in vitro evolution and clinical molecular epidemiology approach. Clinical H3N2 isolates from two sentinel influenza surveillance sites in Lagos and Abuja will be subjected to sequential passage in MDCK cells under escalating oseltamivir concentrations to generate resistant variants. Whole genome sequencing will identify neuraminidase and haemagglutinin mutations acquired during resistance selection. Structural modelling will characterise the impact of identified mutations on neuraminidase active site binding affinity. A retrospective clinical molecular study will screen neuraminidase sequences from 300 archived H3N2-positive samples from 2020 to 2025 for pre-existing resistance-associated substitutions. Phenotypic neuraminidase inhibition assays will confirm resistance in selected clinical isolates. Network analysis will assess whether resistance-carrying strains form phylogenetic clusters suggesting community transmission. This study generates an original mechanistic and evolutionary dataset on oseltamivir resistance in West African H3N2 strains, providing evidence for influenza antiviral stewardship guidelines and the WHO Influenza Antiviral Working Group's surveillance framework. Keywords: oseltamivir resistance, influenza H3N2, neuraminidase, resistance evolution, Nigeria