Brain-derived exosomes differentially regulate pseudorabies virus replication and blood–brain barrier integrity following infection and immunization

Exosomes are critical mediators of virus–host interactions, transferring viral and host components that influence viral replication, immune responses, and tissue injury. Pseudorabies virus (PRV), a neurotropic alphaherpesvirus, disrupts blood–brain barrier (BBB) integrity and causes severe neurological disease, while immunization with the triple-gene-deleted PRV-delgE/gI/TK strain mitigates BBB damage. However, the role of exosomes in PRV-induced neuropathogenesis and vaccine-mediated protection remains unclear. Here, we isolated brain-derived exosomes from PRV-infected, PRV-delgE/gI/TK-immunized and challenged, and mock-treated mice using differential ultracentrifugation combined with CD9/CD63/CD81-based enrichment. Transmission electron microscopy, nanoparticle tracking analysis, and Western blotting confirmed exosome purity and excluded virion contamination. Exosomes from PRV-infected mice contained viral genomic DNA and glycoproteins, whereas those from immunized-and-challenged mice lacked detectable viral components. Functionally, PRV-infected mouse–derived exosomes enhanced PRV replication in PK-15 cells and aggravated BBB disruption in a Transwell-based in vitro BBB model, while exosomes from immunized-and-challenged mice suppressed viral replication and preserved tight junction integrity. Consistently, in vivo administration of immunized-and-challenged mouse–derived exosomes reduced viral load, neurological deficits, and BBB leakage. Collectively, these findings identify brain-derived exosomes as key regulators of PRV replication and BBB integrity and reveal a previously unrecognized mechanism underlying PRV-delgE/gI/TK–mediated neurovascular protection.