Discriminación Rápida de Vías de Entrada de Virus Únicos mediante una Sonda Fluorescente Activable Bioortogonal

Viral transmembrane entry is a critical yet heterogeneous process that governs infectivity and pathogenesis. However, directly distinguishing virus attachment from internalization and resolving distinct entry mechanisms in real time at the single-virus level remains challenging. Here, we introduce a Bioorthogonal Click-Activatable Viral Labeling (CAVL) system for spatiotemporally resolved visualization of viral entry dynamics. This approach combines a membrane-anchored, tetrazine-quenched BODIPY probe (BDP-Tz-MAL) with viral particles dually labeled with trans-cyclooctene (TCO) on their surface and quantum dots (QDs) within their core. Virus-cell contact triggers a rapid inverse electron-demand Diels-Alder (iEDDA) reaction, generating a turn-on fluorescence signal exclusively during transmembrane entry. By integrating this activatable signal with a persistent internal QDs label, we achieve real-time discrimination of endocytic and non-endocytic entry pathways at single-particle resolution. The CAVL platform operates without genetic modification, applies to diverse enveloped and non-enveloped viruses, and provides a quantitative, background-suppressed strategy for elucidating viral entry mechanisms across broad biological contexts.