The Association of Human Astrovirus with Extracellular Vesicles Facilitates Cell Infection and Protects the Virus from Neutralizing Antibodies.

Viral gastroenteritis has a global distribution and represents a high risk for vulnerable population and children under 5 years due to acute diarrhea, fever and dehydration. Human astroviruses (HAstV) have been identified as the third most important cause of viral gastroenteritis in pediatric and immunocompromised patients. Furthermore, HAstV has been reported in biopsies taken from patients with encephalitis, meningitis and acute respiratory infection, yet it is not clear how the virus reaches these organs. In this work we have tested the possibility that the released astrovirus particles could be associated with extracellular vesicles. Comparison between vesicles purified from HAstV Yuc8 infected and mock-infected cells showed that infection enhances production of vesicles larger than 150 nm. These vesicles contain CD63 and Alix, two markers of vesicular structures. Almost 70% of the extracellular virus present in clarified supernatant at 18 h postinfection was found associated with vesicular membranes, and this association facilitates cell infection in the absence of trypsin activation and protects virions from neutralizing antibodies. Our findings suggest a new pathway for HAstV spread and might represent an explanation for the extra-intestinal presence of some astrovirus strains. IMPORTANCE Astroviruses are an important cause of diarrhea in vulnerable population, particularly children; recently some reports have found these viruses in extra-intestinal organs, including the central nervous system, causing unexpected clinical disease. In this work, we found that human astrovirus strain Yuc8 associates with extracellular vesicles, possibly during or after their cell egress. The association with vesicles doubled astrovirus infectivity in less susceptible cells and rendered virus particles insensitive to neutralization by antibodies. These data suggest that extracellular vesicles could represent a novel pathway for astrovirus to disseminate outside the gastrointestinal tract.

Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite, and Immune Cells in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by loss-of-function mutations in the dystrophin gene on chromosome Xp21. Disruption of the dystrophin-glycoprotein complex (DGC) on the cell membrane causes cytosolic Ca2+ influx, resulting in protease activation, mitochondrial dysfunction, and progressive myofiber degeneration, leading to muscle wasting and fragility. In addition to the function of dystrophin in the structural integrity of myofibers, a novel function of asymmetric cell division in muscular stem cells (satellite cells) has been reported. Therefore, it has been suggested that myofiber instability may not be the only cause of dystrophic degeneration, but rather that the phenotype might be caused by multiple factors, including stem cell and myofiber functions. Furthermore, it has been focused functional regulation of satellite cells by intracellular communication of extracellular vesicles (EVs) in DMD pathology. Recently, a novel molecular mechanism of DMD pathogenesis-circulating RNA molecules-has been revealed through the study of target pathways modulated by the Neutral sphingomyelinase2/Neutral sphingomyelinase3 (nSMase2/Smpd3) protein. In addition, adeno-associated virus (AAV) has been clinically applied for DMD therapy owing to the safety and long-term expression of transduction genes. Furthermore, the EV-capsulated AAV vector (EV-AAV) has been shown to be a useful tool for the intervention of DMD, because of the high efficacy of the transgene and avoidance of neutralizing antibodies. Thus, we review application of AAV and EV-AAV vectors for DMD as novel therapeutic strategy.

Deciphering the Role of Extracellular Vesicles Derived from ZIKV-Infected hcMEC/D3 Cells on the Blood-Brain Barrier System.

To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechanisms underlying ZIKV-associated pathogenesis of the central nervous system (CNS) are largely unexplored. Getting more insight into the cellular pathways that ZIKV recruits to facilitate infection of susceptible cells will be crucial for establishing an effective treatment strategy. In general, cells secrete a number of vesicles, known as extracellular vesicles (EVs), in response to viral infections. These EVs serve as intercellular communicators. Here, we investigated the role of EVs derived from ZIKV-infected human brain microvascular endothelial cells on the blood-brain barrier (BBB) system. We demonstrated that ZIKV-infected EVs (IEVs) can incorporate viral components, including ZIKV RNA, NS1, and E-protein, and further transfer them to several types of CNS cells. Using label-free impedance-based biosensing, we observed that ZIKV and IEVs can temporally disturb the monolayer integrity of BBB-mimicking cells, possibly by inducing structural rearrangements of the adherent protein VE-cadherin (immunofluorescence staining). Finally, differences in the lipidomic profile between EVs and their parental cells possibly suggest a preferential sorting mechanism of specific lipid species into the vesicles. To conclude, these data suggest that IEVs could be postulated as vehicles (Trojan horse) for ZIKV transmission via the BBB.

A Comprehensive Analysis of Human Endogenous Retroviruses HERV-K (HML.2) from Teratocarcinoma Cell Lines and Detection of Viral Cargo in Microvesicles.

About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles-MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.

Teriflunomide Inhibits JCPyV Infection and Spread in Glial Cells and Choroid Plexus Epithelial Cells.

Several classes of immunomodulators are used for treating relapsing-remitting multiple sclerosis (RRMS). Most of these disease-modifying therapies, except teriflunomide, carry the risk of progressive multifocal leukoencephalopathy (PML), a severely debilitating, often fatal virus-induced demyelinating disease. Because teriflunomide has been shown to have antiviral activity against DNA viruses, we investigated whether treatment of cells with teriflunomide inhibits infection and spread of JC polyomavirus (JCPyV), the causative agent of PML. Treatment of choroid plexus epithelial cells and astrocytes with teriflunomide reduced JCPyV infection and spread. We also used droplet digital PCR to quantify JCPyV DNA associated with extracellular vesicles isolated from RRMS patients. We detected JCPyV DNA in all patients with confirmed PML diagnosis (n = 2), and in six natalizumab-treated (n = 12), two teriflunomide-treated (n = 7), and two nonimmunomodulated (n = 2) patients. Of the 21 patients, 12 (57%) had detectable JCPyV in either plasma or serum. CSF was uniformly negative for JCPyV. Isolation of extracellular vesicles did not increase the level of detection of JCPyV DNA versus bulk unprocessed biofluid. Overall, our study demonstrated an effect of teriflunomide inhibiting JCPyV infection and spread in glial and choroid plexus epithelial cells. Larger studies using patient samples are needed to correlate these in vitro findings with patient data.

Cocaine augments neuro-inflammation via modulating extracellular vesicle release in HIV-1 infected immune cells.

BACKGROUND: Extracellular Vesicles (EV) recently have been implicated in the pathogenesis of HIV-1 syndromes, including neuroinflammation and HIV-1 associated neurological disorder (HAND). Cocaine, an illicit stimulant drug used worldwide is known to exacerbate these HIV-1 associated neurological syndromes. However, the effects of cocaine on EV biogenesis and roles of EVs in enhancing HIV-1 pathogenesis are not yet well defined. RESULTS: Here, we investigated the effects of cocaine on EV biogenesis and release in HIV-1 infected immune cells and explored their roles in elicitation of neuroinflammation. We found that cocaine significantly augmented the release of EVs from uninfected and HIV-1 infected T-cells, DCs and macrophages. Further analysis of the molecular components of EVs revealed enhanced expression of adhesion molecules integrin ß1 and LFA-1 in those EVs derived from cocaine treated cells. Intriguingly, in EVs derived from HIV-1 infected cells, cocaine treatment significantly increased the levels of viral genes in EVs released from macrophages and DCs, but not in T-cells. Exploring the molecular mechanism to account for this, we found that DCs and macrophages showed enhanced expression of the cocaine receptor Sigma 1-Receptor compared to T-cells. In addition, we found that cocaine significantly altered the integrity of the RNA-induced silencing complex (RISC) in HIV-1 infected macrophages and DCs compared to untreated HIV-1 infected cells. Characterizing further the molecular mechanisms involved in how cocaine increased EV release, we found that cocaine decreased the expression of the interferon-inducible protein BST-2; this resulted in altered trafficking of intracellular virus containing vesicles and EV biogenesis and release. We also observed EVs released from cocaine treated HIV-1 infected macrophages and DCs enhanced HIV-1 trans-infection to T-cells compared to those from untreated and HIV-1 infected cells. These EVs triggered release of proinflammatory cytokines in human brain microvascular endothelial cells (HBMECs) and altered monolayer integrity. CONCLUSIONS: Taken together, our results provide a novel mechanism which helps to elucidate the enhanced prevalence of neurological disorders in cocaine using HIV-1 infected individuals and offers insights into developing novel therapeutic strategies against HAND in these hosts.

Viral Bad News Sent by EVAIL.

This article reviews the current knowledge on how viruses may utilize Extracellular Vesicle Assisted Inflammatory Load (EVAIL) to exert pathologic activities. Viruses are classically considered to exert their pathologic actions through acute or chronic infection followed by the host response. This host response causes the release of cytokines leading to vascular endothelial cell dysfunction and cardiovascular complications. However, viruses may employ an alternative pathway to soluble cytokine-induced pathologies-by initiating the release of extracellular vesicles (EVs), including exosomes. The best-understood example of this alternative pathway is human immunodeficiency virus (HIV)-elicited EVs and their propensity to harm vascular endothelial cells. Specifically, an HIV-encoded accessory protein called the "negative factor" (Nef) was demonstrated in EVs from the body fluids of HIV patients on successful combined antiretroviral therapy (ART); it was also demonstrated to be sufficient in inducing endothelial and cardiovascular dysfunction. This review will highlight HIV-Nef as an example of how HIV can produce EVs loaded with proinflammatory cargo to disseminate cardiovascular pathologies. It will further discuss whether EV production can explain SARS-CoV-2-mediated pulmonary and cardiovascular pathologies.

Plasma Extracellular Vesicles Enhance HIV-1 Infection of Activated CD4+ T Cells and Promote the Activation of Latently Infected J-Lat10.6 Cells via miR-139-5p Transfer.

HIV latency is a challenge to the success of antiretroviral therapy (ART). Hence patients may benefit from interventions that efficiently reactivate the latent virus to be eliminated by ARTs. Here we show that plasma extracellular vesicles (pEVs) can enhance HIV infection of activated CD4+ T cells and reactivate the virus in latently infected J-Lat 10.6 cells. Evaluation of the extravesicular miRNA cargo by a PCR array revealed that pEVs from HIV patients express miR-139-5p. Furthermore, we found that increased levels of miR-139-5p in J-Lat 10.6 cells incubated with pEVs corresponded with reduced expression of the transcription factor, FOXO1. pEV treatment also corresponded with increased miR-139-5p expression in stimulated PD1+ Jurkat cells, but with concomitant upregulation of FOXO1, Fos, Jun, PD-1 and PD-L1. However, J-Lat 10.6 cells incubated with miR-139-5p inhibitor-transfected pEVs from HIV ART-naïve and on-ART patients expressed reduced levels of miR-139-5p than cells treated with pEVs from healthy controls (HC). Collectively, our results indicate that pEV miR-139-5p belongs to a network of miRNAs that can promote cell activation, including latent HIV-infected cells by regulating the expression of FOXO1 and the PD1/PD-L1 promoters, Fos and Jun.

Investigation of Extracellular Vesicles From SARS-CoV-2 Infected Specimens: A Safety Perspective.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, is wreaking havoc around the world. Considering that extracellular vesicles (EVs) released from SARS-CoV-2 infected cells might play a role in a viremic phase contributing to disease progression and that standard methods for EV isolation have been reported to co-isolate viral particles, we would like to recommend the use of heightened laboratory safety measures during the isolation of EVs derived from SARS-CoV-2 infected tissue and blood from COVID-19 patients. Research needs to be conducted to better understand the role of EVs in SARS-CoV-2 infectivity, disease progression, and transmission. EV isolation procedures should include approaches for protection from SARS-CoV-2 contamination. We recommend the EV and virology scientific communities develop collaborative projects where relationships between endogenous EVs and potentially lethal enveloped viruses are addressed to better understand the risks and pathobiology involved.

Extracellular vesicles regulate gap junction-mediated intercellular communication and HIV-1 infection of human neural progenitor cells.

Human immunodeficiency virus-1 (HIV-1) has been shown to cross the blood-brain barrier and cause HIV-associated neurocognitive disorders (HAND) through a process that may involve direct or indirect interactions with the central nervous system (CNS) cells and alterations of amyloid ß (Aß) homeostasis. The present study focused on the mechanisms of HIV-1 infecting human neural progenitor cells (hNPCs) and affecting NPC intercellular communications with human brain endothelial cells (HBMEC). Despite the lack of the CD4 receptor, hNPCs were effectively infected by HIV-1 via a mechanism involving the chemokine receptors, CXCR4 and CCR5. HIV-1 infection increased expression of connexin-43 (Cx43), phosphorylated Cx43 (pCx43), and pannexin 2 (Panx2) protein levels in hNPCs, suggesting alterations in gap-junction (GJ) and pannexin channel communication. Indeed, a functional GJ assay indicated an increase in communication between HIV-infected hNPCs and non-infected HBMEC. We next analyzed the impact of HBMEC-derived extracellular vesicles (EVs) and EVs carrying Aß (EV-Aß) on the expression of Cx43, pCx43, and Panx2 in HIV-1 infected and non-infected hNPCs. Exposure to EV-Aß resulted in significant reduction of Cx43 and pCx43 protein expression in non-infected hNPCs when compared to EV controls. Interestingly, EV-Aß treatment significantly increased levels of Cx43, pCx43, and Panx2 in HIV-1-infected hNPCs when compared to non-infected controls. These results were confirmed in a GJ functional assay and an ATP release assay, which is an indicator of connexin hemichannel and/or pannexin channel functions. Overall, the current study demonstrates the importance of hNPCs in HIV-1 infection and indicates that intercellular communications between infected hNPCs and HBMEC can be effectively modulated by EVs carrying Aß as their cargo.

Extracellular Vesicle TGF-ß1 Is Linked to Cardiopulmonary Dysfunction in Human Immunodeficiency Virus.

Extracellular vesicles (EVs) have emerged as important mediators in cell-cell communication; however, their relevance in pulmonary hypertension (PH) secondary to human immunodeficiency virus (HIV) infection is yet to be explored. Considering that circulating monocytes are the source of the increased number of perivascular macrophages surrounding the remodeled vessels in PH, this study aimed to identify the role of circulating small EVs and EVs released by HIV-infected human monocyte-derived macrophages in the development of PH. We report significantly higher numbers of plasma-derived EVs carrying higher levels of TGF-ß1 (transforming growth factor-ß1) in HIV-positive individuals with PH compared with individuals without PH. Importantly, levels of these TGF-ß1-loaded, plasma-derived EVs correlated with pulmonary arterial systolic pressures and CD4 counts but did not correlate with the Dl CO or viral load. Correspondingly, enhanced TGF-ß1-dependent pulmonary endothelial injury and smooth muscle hyperplasia were observed. HIV-1 infection of monocyte-derived macrophages in the presence of cocaine resulted in an increased number of TGF-ß1-high EVs, and intravenous injection of these EVs in rats led to increased right ventricle systolic pressure accompanied by myocardial injury and increased levels of serum ET-1 (endothelin-1), TNF-α, and cardiac troponin-I. Conversely, pretreatment of rats with TGF-ß receptor 1 inhibitor prevented these EV-mediated changes. Findings define the ability of macrophage-derived small EVs to cause pulmonary vascular modeling and PH via modulation of TGF-ß signaling and suggest clinical implications of circulating TGF-ß-high EVs as a potential biomarker of HIV-associated PH.

Local oncolytic adenovirotherapy produces an abscopal effect via tumor-derived extracellular vesicles.

Extracellular vesicles (EVs) play important roles in various intercellular communication processes. The abscopal effect is an interesting phenomenon in cancer treatment, in which immune activation is generally considered a main factor. We previously developed a telomerase-specific oncolytic adenovirus, Telomelysin (OBP-301), and occasionally observed therapeutic effects on distal tumors after local treatment in immunodeficient mice. In this study, we hypothesized that EVs may be involved in the abscopal effect of OBP-301. EVs isolated from the supernatant of HCT116 human colon carcinoma cells treated with OBP-301 were confirmed to contain OBP-301, and they showed cytotoxic activity (apoptosis and autophagy) similar to OBP-301. In bilateral subcutaneous HCT116 and CT26 tumor models, intratumoral administration of OBP-301 produced potent antitumor effects on tumors that were not directly treated with OBP-301, involving direct mediation by tumor-derived EVs containing OBP-301. This indicates that immune activation is not the main factor in this abscopal effect. Moreover, tumor-derived EVs exhibited high tumor tropism in orthotopic HCT116 rectal tumors, in which adenovirus E1A and adenovirus type 5 proteins were observed in metastatic liver tumors after localized rectal tumor treatment. In conclusion, local treatment with OBP-301 has the potential to produce abscopal effects via tumor-derived EVs.

Emerging roles of extracellular vesicles in COVID-19, a double-edged sword?

The sudden outbreak of SARS-CoV-2-infected disease (COVID-19), initiated from Wuhan, China, has rapidly grown into a global pandemic. Emerging evidence has implicated extracellular vesicles (EVs), a key intercellular communicator, in the pathogenesis and treatment of COVID-19. In the pathogenesis of COVID-19, cells that express ACE2 and CD9 can transfer these viral receptors to other cells via EVs, making recipient cells more susceptible for SARS-CoV-2 infection. Once infected, cells release EVs packaged with viral particles that further facilitate viral spreading and immune evasion, aggravating COVID-19 and its complications. In contrast, EVs derived from stem cells, especially mesenchymal stromal/stem cells, alleviate severe inflammation (cytokine storm) and repair damaged lung cells in COVID-19 by delivery of anti-inflammatory molecules. These therapeutic beneficial EVs can also be engineered into drug delivery platforms or vaccines to fight against COVID-19. Therefore, EVs from diverse sources exhibit distinct effects in regulating viral infection, immune response, and tissue damage/repair, functioning as a double-edged sword in COVID-19. Here, we summarize the recent progress in understanding the pathological roles of EVs in COVID-19. A comprehensive discussion of the therapeutic effects/potentials of EVs is also provided.

Extracellular amoebal-vesicles: potential transmission vehicles for respiratory viruses.

Human respiratory syncytial virus (RSV) is a major cause of acute respiratory tract infections in children and immunocompromised adults worldwide. Here we report that amoebae-release respirable-sized vesicles containing high concentrations of infectious RSV that persisted for the duration of the experiment. Given the ubiquity of amoebae in moist environments, our results suggest that extracellular amoebal-vesicles could contribute to the environmental persistence of respiratory viruses, including potential resistance to disinfection processes and thereby offering novel pathways for viral dissemination and transmission.

Extracellular vesicles from HTLV-1 infected cells modulate target cells and viral spread.

BACKGROUND: The Human T-cell Lymphotropic Virus Type-1 (HTLV-1) is a blood-borne pathogen and etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 has currently infected up to 10 million globally with highly endemic areas in Japan, Africa, the Caribbean and South America. We have previously shown that Extracellular Vesicles (EVs) enhance HTLV-1 transmission by promoting cell-cell contact. RESULTS: Here, we separated EVs into subpopulations using differential ultracentrifugation (DUC) at speeds of 2 k (2000×g), 10 k (10,000×g), and 100 k (100,000×g) from infected cell supernatants. Proteomic analysis revealed that EVs contain the highest viral/host protein abundance in the 2 k subpopulation (2 k > 10 k > 100 k). The 2 k and 10 k populations contained viral proteins (i.e., p19 and Tax), and autophagy proteins (i.e., LC3 and p62) suggesting presence of autophagosomes as well as core histones. Interestingly, the use of 2 k EVs in an angiogenesis assay (mesenchymal stem cells + endothelial cells) caused deterioration of vascular-like-tubules. Cells commonly associated with the neurovascular unit (i.e., astrocytes, neurons, and macrophages) in the blood-brain barrier (BBB) showed that HTLV-1 EVs may induce expression of cytokines involved in migration (i.e., IL-8; 100 k > 2 k > 10 k) from astrocytes and monocyte-derived macrophages (i.e., IL-8; 2 k > 10 k). Finally, we found that EVs were able to promote cell-cell contact and viral transmission in monocytic cell-derived dendritic cell. The EVs from both 2 k and 10 k increased HTLV-1 spread in a humanized mouse model, as evidenced by an increase in proviral DNA and RNA in the Blood, Lymph Node, and Spleen. CONCLUSIONS: Altogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Carrying miRNA as a Potential Multi Target Therapy to COVID-19: an In Silico Analysis.

In the end of 2019 COVID-19 emerged as a new threat worldwide and this disease present impaired immune system, exacerbated production of inflammatory cytokines, and coagulation disturbs. Mesenchymal stem cell (MSC) derived extracellular vesicles (EVs) have emerged as a therapeutic option due to its intrinsic properties to alleviate inflammatory responses, capable to promote the restoring of injured tissue. EVs contain heterogeneous cargo, including active microRNAs, small noncoding sequences involved in post-transcriptional gene repression or degradation and can attach in multiple targets. This study investigated whether the MSC-EVs miRNA cargo has the capacity to modulate the exacerbated cytokines, cell death and coagulation disturbs present in severe COVID-19. Through bioinformatics analysis, four datasets of miRNA, using different stem cell tissue sources (bone marrow, umbilical cord and adipose tissue), and one dataset of mRNA (bone marrow) were analyzed. 58 miRNAs overlap in the four miRNA datasets analyzed. Sequentially, those miRNAs present in at least two datasets, were analyzed using miRWalk for the 3'UTR binding target mRNA. The result predicted 258 miRNAs for exacerbated cytokines and chemokines, 266 miRNAs for cell death genes and 148 miRNAs for coagulation cascades. Some miRNAs may simultaneously attenuate inflammatory agents, inhibit cell death genes and key factors of coagulation cascade, consequently preventing tissue damage and coagulation disturbs. Therefore, the MSC-derived EVs due to their heterogeneous cargo are a potential multitarget approach able to improve the survival rates of severe COVID-19 patients.

Kaposi's sarcoma-associated herpesvirus and extracellular vesicles.

Kaposi's sarcoma-associated herpesvirus (KSHV) represents the etiological agent for several human malignancies, including Kaposi's Sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), which develop mainly in immunocompromised patients. KSHV has established many strategies to hijack and thwart the host's immune responses, including through the use of extracellular vesicles (EVs). EVs represent a significant mode of intercellular communication as they carry a variety of molecules that can be delivered from cell-to-cell. EVs are now recognized as one of the major players in immune system development and function during both innate and adaptive immune responses. In the current mini-review, we summarize recent findings on how KSHV utilizes EVs to create favorable environments for viral spread and persistence while evading immune responses. We also discuss the limitations and unanswered questions in this field and the potential areas for related immunotherapies.

Emerging Role of Nef in the Development of HIV Associated Neurological Disorders.

Despite adherence to treatment, individuals living with HIV have an increased risk for developing cognitive impairments, referred to as HIV-associated neurological disorders (HAND). Due to continued growth in the HIV population, particularly amongst the aging cohort, the neurobiological mechanisms of HAND are increasingly relevant. Similar to other viral proteins (e.g. Tat, Gp120, Vpr), the Negative Factor (Nef) is associated with numerous adverse effects in the CNS as well as cognitive impairments. In particular, emerging data indicate the consequences of Nef may be facilitated by the modulation of cellular autophagy as well as its inclusion into extracellular vesicles (EVs). The present review examines evidence for the molecular mechanisms by which Nef might contribute to neuronal dysfunction underlying HAND, with a specific focus on autophagy and EVs. Based on the these data, we propose an integrated model by which Nef may contribute to underlying neuronal dysfunction in HAND and highlight potentially novel therapeutic targets for HAND. Graphical abstract.

Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles.

Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus-removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)-containing plasma by a combination of size-exclusion chromatography and affinity-based purification. After purification, EV samples were free of virus-sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential.