Human enterovirus 71 (EV71) is one of the major agents of the hand, foot, and mouth disease (HFMD), and occasionally causes severe neurological complications. There is clinical evidence that EV71 infection increases the exosomes in the serum of severe HFMD patients, suggesting a role of exosomes in EV71 pathogenesis. However, the relationship between exosomes and EV71 replication remains elusive. In this study, we initially found that EV71 infection elevated exosome biogenesis in the cultured cells. Among EV71 non-structural proteins, we identified EV71 3A, but not 3B, constitutively promoted exosome secretion. In detail, EV71 3A protein interacted with vacuolar protein sorting 25 (VPS25), while knock-down of VPS25 reduced EV71 3A protein- and EV71-induced exosome production. Further studies revealed VPS25 located on exosomes and its expression correlated to the exosome production. During EV71 infection, knock-down of VPS25 decreased exosome biogenesis to attenuate viral replication. Consistently, GW4869, an exosome inhibitor, exerted an obviously antiviral activity against EV71 replication companied with the decrease of exosome secretion or formation. These findings suggest the binding of EV71 3A and VPS25 benefited exosome biogenesis, thereby boosting viral replication. This study uncovers a novel mechanism underlying EV71-mediated exosomes in the regulation of viral replication, which provides potential anti-viral strategies against the EV71 infection and transmission in HFMD.

The oxidative stress induced by the accumulation of reactive oxygen species (ROS) can lead to cell aging and death. Equally, the skeletal muscle usually hosts enteroviral persistent infection in inflammatory muscle diseases. As excellent bioactive products, the exosomes derived from umbilical cord mesenchymal stem cells (ucMSCs) have been proven to be safe and have low immunogenicity with a potential cell-free therapeutic function. Here, exosomes derived from ucMSCs (ucMSC-EXO) were extracted and characterized. In a model of oxidative damage to skin fibroblasts (HSFs) under exposure to H₂O₂, ucMSC-EXO had an observable repairing effect for the HSFs suffering from oxidative damage. Furthermore, ucMSC-EXO inhibited mitogen-activated protein kinases (MAPK), c-Jun N-terminal kinase (JNK), and nuclear factor kappa-B (NF-κB) signaling pathways, thereby promoting p21 protein expression while decreasing lamin B1 protein expression, and finally alleviated oxidative stress-induced cell damage and aging. In a model of rhabdomyosarcoma (RD) cells being infected by enterovirus 71 (EV71) and coxsackievirus B3 (CVB3), the ucMSC-EXO enhanced the expression of interferon-stimulated gene 15 (ISG15) and ISG56 to inhibit enteroviral replication, whereafter reducing the virus-induced proinflammatory factor production. This study provides a promising therapeutic strategy for ucMSC-EXO in anti-oxidative stress and antiviral effects, which provides insight into extending the function of ucMSC-EXO in cell-free therapy.

Enterovirus A71 infection can cause hand, foot, and mouth disease to occasionally develop the severe neurological complications, but how peripheral Enterovirus A71 infection affects the blood-brain barrier remains incompletely understood. Here, we show that Enterovirus A71 infection of peripheral susceptible cells increases the release of extracellular vesicles and promotes the enrichment of microRNA-30 and microRNA-34 within these vesicles. The extracellular vesicle-associated microRNAs suppress Notch1 signaling in human brain microvascular endothelial cells, impair endothelial junctional integrity, and increase blood-brain barrier permeability. Activation of Notch1 signaling restores barrier integrity and reduces viral neuroinvasion in neonatal mice. Clinically, serum extracellular vesicle-associated microRNA-30 and microRNA-34 are elevated in patients with severe Enterovirus A71-associated hand, foot, and mouth disease. Together, our findings identify circulating extracellular vesicles as mediators linking peripheral Enterovirus A71 infection to blood-brain barrier dysfunction and suggest that the microRNA-30/34–Notch1 axis contributes to neurological disease progression during severe Enterovirus A71 infection.