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Key Points The transfer of extracellular vesicles (EVs) from one cell to another is thought to be an important mechanism for cell–cell communication, and EVs that are produced by virus-infected cells may modulate uninfected cells. Viruses have specific receptors and therefore usually have a more restricted cellular tropism. By contrast, the uptake of EVs is almost universal and can occur through several cellular endocytic mechanisms in addition to direct fusion, a property that enables systemic delivery of their content. The differences between viral and EV receptor usage can be used to separate and purify EVs. This enables the identification of specific EV-mediated effects and enables compounds that potentially inhibit the delivery and function of extracellular vesicles to be tested. Viruses that establish chronic and persistent infections in a host probably use EVs to enhance the establishment and maintenance of infection. The EVs that are produced from virus-infected cells (and may therefore differ in content) may also restrict virus infection and enable continued host viability and persistent viral infection. The incorporation of virions into EVs could prevent the recognition of viral proteins by the immune system and facilitate spread in the host. The release of membrane-bound vesicles from cells is being increasingly recognized as a mechanism of intercellular communication. In this Review, Raab-Traub and Dittmer discuss the roles that extracellular vesicles have during virus infection. The release of membrane-bound vesicles from cells is being increasingly recognized as a mechanism of intercellular communication. Extracellular vesicles (EVs) or exosomes are produced by virus-infected cells and are thought to be involved in intercellular communication between infected and uninfected cells. Viruses, in particular oncogenic viruses and viruses that establish chronic infections, have been shown to modulate the production and content of EVs. Viral microRNAs, proteins and even entire virions can be incorporated into EVs, which can affect the immune recognition of viruses or modulate neighbouring cells. In this Review, we discuss the roles that EVs have during viral infection to either promote or restrict viral replication in target cells. We will also discuss our current understanding of the molecular mechanisms that underlie these roles, the potential consequences for the infected host and possible future diagnostic applications.

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy (PML) in immunosuppressed and immunomodulated patients. Initial infection with JCPyV is common and the virus establishes a long-term persistent infection in the urogenital system of 50-70% of the human population worldwide. A major gap in the field is that we do not know how the virus traffics from the periphery to the brain to cause disease. Our recent discovery that human choroid plexus epithelial cells are fully susceptible to virus infection together with reports of JCPyV infection of choroid plexus in vivo has led us to hypothesize that the choroid plexus plays a fundamental role in this process. The choroid plexus is known to relay information between the blood and the brain by the release of extracellular vesicles. This is particularly important because human macroglia (oligodendrocytes and astrocytes), the major targets of virus infection in the central nervous system (CNS), do not express the known attachment receptors for the virus and do not bind virus in human tissue sections. In this report we show that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles that contain JCPyV and readily transmit the infection to human glial cells. Transmission of the virus by extracellular vesicles is independent of the known virus attachment receptors and is not neutralized by antisera directed at the virus. We also show that extracellular vesicles containing virus are taken into target glial cells by both clathrin dependent endocytosis and macropinocytosis. Our data support the hypothesis that the choroid plexus plays a fundamental role in the dissemination of virus to brain parenchyma.

Viral spread by both enveloped and non-enveloped viruses may be mediated by extracellular vesicles (EVs), including microvesicles (MVs) and exosomes. These secreted vesicles have been demonstrated to be an efficient mechanism that viruses can use to enter host cells, enhance spread or evade the host immune response. However, the complex interplay between viruses and EVs gives rise to antagonistic biological tasks—to benefit the viruses, enhancing infection and interfering with the immune system or to benefit the host, by mediating anti-viral responses. Exosomes from cells infected with herpes simplex type 1 (HSV-1) may transport viral and host transcripts, proteins and innate immune components. This virus may also use MVs to expand its tropism and evade the host immune response. This review aims to describe the current knowledge about EVs and their participation in viral infection, with a specific focus on the role of exosomes and MVs in herpesvirus infections, particularly that of HSV-1.

Emerging evidence indicates important interconnections between autophagy and the secretion of small extracellular vesicles. However, our understanding of these secretory vesicles remains incomplete. Here, we identify a subtype of small extracellular vesicles, termed autophagic extracellular vesicles (AEVs), which are distinct from exosomes. Extracellular AEVs characterized by a size less than 100 nm exhibit increased secretion when the autophagy response is induced. Amphisomes, the hybrid organelles, are essential for the secretion of these types of vesicles. Further exploration reveals that autophagic cargos, certain ESCRT Ⅲ components and the Rab13 serve as distinctive markers for distinguishing AEVs from exosomes. Moreover, we find that the biogenesis of AEVs functionally requires components of the ESCRT Ⅲ complex and the GTPase Rab27a. Finally, we confirm that the enteroviral particles or genomes can be encapsulated into AEVs and subsequently infect receptor-negative cells with high efficiency. This model represents a prominent pattern of EV-mediated virus transmission. The authors identify a subtype of small extracellular vesicles, termed autophagic extracellular vesicles. This type of vesicle exhibits biomarkers different from exosomes and plays a crucial role in viral infections.

Coxsackievirus B3 (CVB3) is a picornavirus that causes systemic inflammatory diseases including myocarditis, pericarditis, pancreatitis, and meningoencephalitis. We have previously reported that CVB3 induces mitochondrial fission and mitophagy while inhibiting lysosomal degradation by blocking autophagosome-lysosome fusion. This promotes the release of virus-laden mitophagosomes from host cells as infectious extracellular vesicles (EVs), enabling non-lytic viral egress. Transient receptor potential vanilloid 1 (TRPV1), a heat and capsaicin-sensitive cation channel, regulates mitochondrial dynamics by inducing mitochondrial membrane depolarization and fission. In this study, we found that TRPV1 activation by capsaicin dramatically enhances CVB3 egress from host cells via EVs. Released EVs revealed increased levels of viral capsid protein VP1, mitochondrial protein TOM70, and fission protein phospho-DRP1. Moreover, these EVs were enriched in heat shock protein HSP70, suggesting its role in facilitating infectious EV release from cells. Furthermore, TRPV1 inhibition with capsazepine and SB-366791 significantly reduced viral infection in vitro. Our in vivo studies also found that SB-366791 significantly mitigates pancreatic damage and reduces viral titers in a mouse model of CVB3 pancreatitis. Given the lack of understanding regarding factors that contribute to diverse clinical manifestations of CVB3, our study highlights capsaicin and TRPV1 as potential exacerbating factors that facilitate CVB3 dissemination via mitophagy-derived EVs.

Bacteria and phages have coexisted for billions of years engaging in continuous evolutionary arms races that drive reciprocal adaptations and resistance mechanisms. Among the diverse antiviral strategies developed by bacteria, modification or masking phage receptors as well as their physical removal via extracellular vesicles are the first line of defense. These vesicles play a pivotal role in bacterial survival by mitigating the effects of various environmental threats, including predation by bacteriophages. The secretion of extracellular vesicles represents a highly conserved evolutionary trait observed across all domains of life. Bacterial extracellular vesicles (BEVs) are generated by a wide variety of Gram (+), Gram (−), and atypical bacteria, occurring under both natural and stress conditions, including phage infection. This review addresses the multifaceted role of BEVs in modulating bacteria–phage interactions, considering the interplay from both bacterial and phage perspectives. We focus on the dual function of BEVs as both defensive agents that inhibit phage infection and as potential facilitators that may inadvertently enhance bacterial susceptibility to phages. Furthermore, we discuss how bacteriophages can influence BEV production, affecting both the quantity and molecular composition of vesicles. Finally, we provide an overview of the ecological relevance and efficacy of BEV–phage interplay across diverse environments and microbial ecosystems.

Bluetongue virus (BTV) is one of the most economically relevant orbiviruses and is the only example of a large complex, but non-enveloped arbovirus. In addition to cell lysis, BTV is known to employ a ‘budding’ process analogous to that used by enveloped viruses for cell exit, in which the viral glycosylated NS3 protein plays a key role. Recent reports have demonstrated that BTV can also induce non-lytic release via extracellular vesicles (EVs), however, details of the type and origin of the EV used and the role of NS3 in the process remain incompletely understood. In this study we undertook biochemical studies on the non-lytic release of BTV particles in different forms of EVs from several types of host cells and complemented this by comprehensive microscopic analyses using fluorescence microscopy, transmission electron microscopy and electron cryo-tomography. We discovered that BTV particles use both large EVs (LEVs) and smaller size EVs (SEVs) for non-lytic release and that, in each cell type studied, SEV fractions were particularly enriched with NS3. Non-enveloped BTV particles initially released in SEVs were highly infectious and promote efficient cell-to-cell transmission. This discovery highlights the complex mechanisms utilized by a non-enveloped arbovirus for egress and the significance of different EV types in this process.

Zika virus (ZIKV) is a neurotropic flavivirus linked to severe neurodevelopmental defects following prenatal exposure. While the mechanisms by which ZIKV spreads within the central nervous system remain incompletely understood, extracellular vesicles (EVs) have emerged as potential mediators of intercellular communication and viral dissemination. Here, we demonstrate that EVs derived from ZIKV-infected neural cells encapsulate full-length viral genomes capable of establishing productive in vivo infection, independent of free virions. Primary cortical neurons, astrocytes, and mouse brain microvascular endothelial cells (MBECs) from neonatal mice were infected with ZIKV at a low multiplicity of infection (MOI 0.1). EVs were isolated and treated with acid glycine buffer and RNAase to exclude residual virions or free RNA. RNA sequencing, RT-qPCR, and droplet digital PCR (dd-PCR) analyses revealed that EVs—particularly those derived from neurons and MBECs—encapsulated ZIKV RNA, including full-length viral genomes. These EVs were able to transfer viral RNA to A549 cells in vitro , and its intracranial injection into neonatal mice resulted in productive infection, confirmed by detection of ZIKV capsid protein, viral RNA, and viral antigen in brain tissue. Our findings demonstrate that EVs from ZIKV-infected neural cells can serve as vehicles for genome transfer and initiate infection, even in the absence of detectable virions. The persistence of EVs-packaged genomes post-viremia could explain clinical observations of prolonged ZIKV RNA within the nervous tissue or delayed transmission. Understanding this pathway provides new insights into ZIKV neuropathogenesis and opens potential avenues for therapeutic intervention, for example targeting EVs biogenesis or cargo sorting.

Hepatitis B virus (HBV) generates large amounts of complete and incomplete viral particles. Except for the virion, which acts as infectious particles, the function of those particles remains elusive. Extracellular vesicles (EVs) have been revealed to have biological functions. The EVs which size are less than 100 nm in diameter, were collected from HBV infected-patients. These vesicles contain, complete and incomplete virions, and exosomes, which have been recently shown to be critical as intercellular communicators. Here, the effects of the exosome, the complete, and the incomplete particles on the target cells were investigated. These particles are endocytosed by monocyte/macrophages and function primarily to upregulate PD-L1. The functions and composition of the EVs were affected by nucleotide reverse transcriptase inhibitors (NRTIs), suggesting that the EVs are involved in the pathogenesis of HBV hepatitis and clinical course of those patients treated by NRTIs.

Although neurological disease is common in people with human immunodeficiency virus (HIV) (PWH), the contributing factors and underlying inflammatory mechanisms remain challenging to identify. Extracellular vesicles (EVs) constitute a relatively uncharacterized modality of intercellular communication and bioactive cargo transport in the setting of viral infection and pathogenesis. EVs carry inflammatory mediators to areas of the periphery during antiretroviral therapy (ART) suppression but are understudied in the brain. Using a biologically relevant simian–human immunodeficiency chimeric virus with a clade D HIV envelope (SHIV.D)-infected rhesus macaque (RM) model of HIV persistence in the central nervous system (CNS), we investigate circulating EV populations and the protein cargo of myeloid-derived EVs during SHIV infection. Using EV flow cytometry to quantify specific EV subpopulations, we found a significant increase in TMEM119+ microglial EVs and CD171+ neuronal EVs in RM plasma during viremia and ART suppression. Using primary RM monocyte-derived macrophages (MDMs), we determined that MDMs increased EV production after SHIV infection. Whole proteomic analysis of these EVs demonstrated that myeloid EVs isolated from SHIV.D-infected MDMs carried significantly increased levels of neuropathogenic and inflammatory proteins. Altogether, these studies improve our understanding of the contribution of myeloid EVs to neurological disease during SHIV/HIV infection.