Integrated Single Nanoparticle Analysis for Rapid Quantification of Spatiotemporal Crosstalk between Herpes Simplex Virus‑1 and Extracellular Vesicles

Cells secrete extracellular vesicles (EVs) to mediate precise communication during viral infections, yet the spatiotemporal regulation of EV composition by herpes simplex virus 1 (HSV-1) remains poorly understood. Here, we develop an integrated single-nanoparticle analysis platform combining nanoporous membrane-based EV isolation with an on-chip immunoassay to quantitatively probe EV–HSV-1 interplay throughout infection. A dual-membrane filter design significantly enhances nanoparticle recovery, enabling high-sensitivity single-particle detection. We reveal that HSV-1-infected neural stem cells display viral glycoprotein B on EV surfaces at an early stage (<8 hpi), while intact virions are selectively packaged into EVs later (24–48 hpi). Proteomic profiling indicates infected cell-derived EVs facilitate antigen processing and presentation, potentially amplifying antiviral responses. Functional studies further demonstrate EVs promote viral entry at late stages (48 hpi), likely via EV-virion encapsulation. These findings elucidate a dynamic EV–virus interplay, offering insights into HSV-1 pathogenesis and EV-mediated immune modulation. Our platform provides a transformative approach for advancing infection diagnostics and therapeutics.