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.