Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

Life stage-specific glycosylation of extracellular vesicles from Schistosoma mansoni schistosomula and adult worms drives differential interaction with C-type lectin receptors DC-SIGN and MGL.

Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells via C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000 nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcß1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy.

Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a 'viral security protein' that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs.

Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield.

In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources.

CDC6: A novel canine tumour biomarker detected in circulating extracellular vesicles.

Circulating nucleic acids and extracellular vesicles (EV) represent novel biomarkers to diagnose cancer. The non-invasive nature of these so-called liquid biopsies provides an attractive alternative to tissue biopsy-based cancer diagnostics. This study aimed to investigate if circulating cell cycle-related E2F target transcripts can be used to diagnose tumours in canine tumour patients with different types of tumours. Furthermore, we assessed if these mRNAs are localised within circulating EV. We isolated total RNA from the plasma of 20 canine tumour patients and 20 healthy controls. Four E2F target genes (CDC6, DHFR, H2AFZ and ATAD2) were selected based on the analysis of published data of tumour samples available in public databases. We performed reverse transcription and quantitative real-time PCR to analyse the plasma levels of selected E2F target transcripts. All four E2F target transcripts were detectable in the plasma of canine tumour patients. CDC6 mRNA levels were significantly higher in the plasma of canine tumour patients compared to healthy controls. A subset of canine tumour patient and healthy control plasma samples (n = 7) were subjected to size exclusion chromatography in order to validate association of the E2F target transcripts to circulating EV. For CDC6, EV analysis enhanced their detectability compared to total plasma analysis. In conclusion, our study reveals circulating CDC6 as a promising non-invasive biomarker to diagnose canine tumours.

The power of imaging to understand extracellular vesicle biology in vivo.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

Malaria parasites both repress host CXCL10 and use it as a cue for growth acceleration.

Pathogens are thought to use host molecular cues to control when to initiate life-cycle transitions, but these signals are mostly unknown, particularly for the parasitic disease malaria caused by Plasmodium falciparum. The chemokine CXCL10 is present at high levels in fatal cases of cerebral malaria patients, but is reduced in patients who survive and do not have complications. Here we show a Pf 'decision-sensing-system' controlled by CXCL10 concentration. High CXCL10 expression prompts P. falciparum to initiate a survival strategy via growth acceleration. Remarkably, P. falciparum inhibits CXCL10 synthesis in monocytes by disrupting the association of host ribosomes with CXCL10 transcripts. The underlying inhibition cascade involves RNA cargo delivery into monocytes that triggers RIG-I, which leads to HUR1 binding to an AU-rich domain of the CXCL10 3'UTR. These data indicate that when the parasite can no longer keep CXCL10 at low levels, it can exploit the chemokine as a cue to shift tactics and escape.

miREV: An Online Database and Tool to Uncover Potential Reference RNAs and Biomarkers in Small-RNA Sequencing Data Sets from Extracellular Vesicles Enriched Samples.

Extracellular vesicles (EVs) are nano-sized, membrane-enclosed vesicles released by cells for intercellular communication. EVs are involved in pathological processes and miRNAs in EVs have gained interest as easily accessible biomolecules in liquid biopsies for diagnostic purposes. To validate potential miRNA biomarker, transcriptome analyses must be carried out to detect suitable reference miRNAs. miREV is a database with over 400 miRNA sequencing data sets and helps the researcher to find suitable reference miRNAs for their individual experimental setup. The researcher can put together a specific sample set in miREV, which is similar to his own experimental concept in order to find the most suitable references. This allows to run validation experiments without having to carry out a complex and costly transcriptome analysis priorly. Additional read count tables of each generated sample set are downloadable for further analysis. miREV is freely available at https://www.physio.wzw.tum.de/mirev/.

Extracellular Vesicles: Novel Opportunities to Understand and Detect Neoplastic Diseases.

With a size range from 30 to 1000 nm, extracellular vesicles (EVs) are one of the smallest cell components able to transport biologically active molecules. They mediate intercellular communications and play a fundamental role in the maintenance of tissue homeostasis and pathogenesis in several types of diseases. In particular, EVs actively contribute to cancer initiation and progression, and there is emerging understanding of their role in creation of the metastatic niche. This fact underlies the recent exponential growth in EV research, which has improved our understanding of their specific roles in disease and their potential applications in diagnosis and therapy. EVs and their biomolecular cargo reflect the state of the diseased donor cells, and can be detected in body fluids and exploited as biomarkers in cancer and other diseases. Relatively few studies have been published on EVs in the veterinary field. This review provides an overview of the features and biology of EVs as well as recent developments in EV research including techniques for isolation and analysis, and will address the way in which the EVs released by diseased tissues can be studied and exploited in the field of veterinary pathology. Uniquely, this review emphasizes the important contribution that pathologists can make to the field of EV research: pathologists can help EV scientists in studying and confirming the role of EVs and their molecular cargo in diseased tissues and as biomarkers in liquid biopsies.

Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses.

Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract.

Integrins Control Vesicular Trafficking; New Tricks for Old Dogs.

Integrins are transmembrane receptors that transduce biochemical and mechanical signals across the plasma membrane and promote cell adhesion and migration. In addition, integrin adhesion complexes are functionally and structurally linked to components of the intracellular trafficking machinery and accumulating data now reveal that they are key regulators of endocytosis and exocytosis in a variety of cell types. Here, we highlight recent insights into integrin control of intracellular trafficking in processes such as degranulation, mechanotransduction, cell-cell communication, antibody production, virus entry, Toll-like receptor signaling, autophagy, and phagocytosis, as well as the release and uptake of extracellular vesicles. We discuss the underlying molecular mechanisms and the implications for a range of pathophysiological contexts, including hemostasis, immunity, tissue repair, cancer, and viral infection.

Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients.

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases.

Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific "Y-RNA signatures" in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases.

Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection.

The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.

DC-SIGN mediated internalisation of glycosylated extracellular vesicles from Schistosoma mansoni increases activation of monocyte-derived dendritic cells.

Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galß1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation.

The generation and use of recombinant extracellular vesicles as biological reference material.

Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications.

Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential.

Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50-300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses.

Technical approaches to reduce interference of Fetal calf serum derived RNA in the analysis of extracellular vesicle RNA from cultured cells.

Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contaminating extracellular vesicles (EV) containing RNA. Because of a high degree of sequence similarity among homologous non-coding RNAs of mammalian species, residual FCS-RNA in culture medium may interfere in the analysis of EV-RNA released by cultured cells. Recently, doubts have been raised as to whether commonly used protocols for depletion of FCS-EV efficiently remove FCS-RNA. Moreover, technical details in FCS-EV depletion protocols are known to vary between labs, which may lead to inter-study differences in contaminating FCS-RNA levels. Here, we investigated how technical modifications of EV-depletion protocols affect the efficiency with which bovine RNAs are depleted from FCS, and determined the contribution of contaminating bovine RNA to EV-RNA purified from cell cultures. Our data show differences in depletion efficiency between and within various classes of small non-coding RNA. Importantly, we demonstrate that variations in FCS-EV depletion protocols affect both the quantity and type of residual FCS-RNAs in EV-depleted medium. By using optimised FCS-EV depletion protocols combined with methods for high-grade purification of EV the levels of contaminating bovine RNA in EV populations isolated from cell culture medium can be reduced. With illustrative datasets we also demonstrate that the abundance of a specific RNA in cell culture EV can only be determined if measured relative to background levels of this RNA in medium control samples. These data highlight the need for optimisation and validation of existing and novel FCS-EV depletion methods and urge for accurate descriptions of these methods in publications to increase experimental reproducibility.