Opportunistic untargeted sequencing can complement targeted viral surveillance workflows for unresolved respiratory illness in Uganda

Abstract Routine respiratory surveillance in low- and middle-income countries relies heavily on targeted molecular diagnostics for influenza and SARS-CoV-2, leaving a substantial proportion of influenza-like illness (ILI) cases etiologically unresolved. To address this diagnostic blind spot, we evaluated the utility of opportunistic, untargeted shotgun next-generation sequencing of viral RNA on a small cluster of four unresolved clinical samples collected in Uganda in December 2025 that had tested negative via routine RT-qPCR. This opportunistic sequencing approach successfully identified clinically relevant viral pathogens in two of the four cases. From one sample, a near-complete genome of Human Metapneumovirus was recovered (13,273 bp, 45.6× coverage). This genome belonged to the globally circulating B2 lineage and revealed greater sequence divergence in the attachment G gene relative to the rest of the genome. The second sample yielded a 368 bp partial fragment of Human Respirovirus 1. These findings demonstrate that the opportunistic application of untargeted sequencing to diagnostically unresolved ILI cases can effectively complement targeted viral workflows, offering a robust, periodic tool to uncover missed pathogens in respiratory viral surveillance.