Rapid Discrimination of Single‐Virus Entry Pathways via a Bioorthogonal Activatable Fluorescent Probe

ABSTRACT 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.