Modern Techniques for the Isolation of Extracellular Vesicles and Viruses

Abstract

Extracellular signaling is pivotal to maintain organismal homeostasis. A quickly emerging field of interest within extracellular signaling is the study of extracellular vesicles (EV), which act as messaging vehicles for nucleic acids, proteins, metabolites, lipids, etc. from donor cells to recipient cells. This transfer of biologically active material within a vesicular body is similar to the infection of a cell through a virus particle, which transfers genetic material from one cell to another to preserve an infection state, and viruses are known to modulate EV. Although considerable heterogeneity exists within EV and viruses, this review focuses on those that are small (< 200 nm in diameter) and of relatively low density (< 1.3 g/mL). A multitude of isolation methods for EV and virus particles exist. In this review, we present an update on methods for their isolation, purification, and phenotypic characterization. We hope that the information we provide will be of use to basic science and clinical investigators, as well as biotechnologists in this emerging field.

Graphical Abstract

Introduction

Extracellular vesicles (EV) are secreted from every cell type studied. Several subtypes of EV exist and are classified based on their sizes, cellular origin and surface markers. Apoptotic bodies (800–5000 nm in diameter) and microvesicles (100–800 nm in diameter) bud off at the plasma membrane and are thus enriched for proteins sitting on the cell surface. Exosomes are a class of EV of a small diameter (40–150 nm in diameter) that originate from the inward budding of endosomes into the multivesicular body and contain endosomal trafficking markers such as tumor susceptibility gene 101 (Tsg101), apoptosis-linked gene-2 interacting protein X (Alix), tetraspanins, and flotillins (reviewed in (Crescitelli et al. 2013; Raab-Traub and Dittmer 2017)). EV have received a substantial amount of attention in recent years. They act as an extracellular mailing system, transferring information from one cell to the next, and can contain nucleic acids such as DNA, micro RNAs (miRNA) and non-coding RNAs (ncRNA), mRNA, proteins and enzymes such as histones and esterases, metabolites, and lipids (Chugh et al. 2013; Hurwitz et al. 2016; Meckes et al. 2010, 2013; Wang et al. 2015; Willms et al. 2016; Bukong et al. 2014; Longatti et al. 2015). EV have been proposed to play a critical role in cell differentiation, angiogenesis, metabolic reprogramming, tumor progression, immune modulation, and response to pathogen challenge (Willms et al. 2018; McNamara et al. 2018b; Raab-Traub and Dittmer 2017; Baranyai et al. 2015; Chevillet et al. 2014; M. R. Anderson et al. 2016).

Here, we will review recent updates on the topic of EV isolation using the field of virus isolation for comparison. Virus isolation has been the focus of scientific and engineering studies for well over 100 years, with a particular emphasis on vaccine manufacturing (reviewed in (Effio and Hubbuch 2015). Thus virus isolation represents a validated catalog of tools against which EV isolation techniques can be compared to. Specifically, we will review large-scale methods of ultracentrifugation, precipitation with crowding reagents, crossflow filtration, affinity purification, and nanoscale flow cytometry, and also summarize methods for characterization. Of note, the international society for extracellular vesicles (ISEV) regularly publishes best practices for the field (Théry et al. 2018). These will complement this review.

In human and non-human primate plasma, the concentration of EV is believed to be as high as 1011 particles/mL (McNamara et al. 2018a, b; Chevillet et al. 2014; Baranyai et al. 2015). Even though they are similar in size, these small circulating EV far outnumber even high-titer bloodborne viruses such as West Nile virus, HIV, and Ebola virus during infection. Concentrations of EV < 150 nm in cell culture supernatant also are much higher than in vitro propagated viruses and live-attenuated vaccines or viral like particles (Fig. 1). It has been proposed that every cell type is capable of releasing EV, irrespective of how quickly it divides (Théry et al. 2018; Witwer et al. 2013). To that end, EV have been isolated from various human fluids such as plasma, urine, tumor fluid, effusions, and cerebrospinal fluid. While specific contents can vary depending on cell origin, most EV < 150 nm contain core constituents: (I) tetraspanin proteins, most notably CD9, CD63 and CD81, (II) a lipid membrane rich in phosphatidyl serine, (III) and trafficking proteins such as endosomal sorting complexes required for intracellular transport (ESCRT) machinery (Raab-Traub and Dittmer 2017; Anderson et al. 2016).

Fig. 1
figure1

Comparison of non-enveloped viruses, enveloped viruses, and EV. Transmission electron micrographs of a non-enveloped virus (West Nile virus), an enveloped virus (Simian immunodeficiency virus), and EV are shown. Compositions of the particles, such as capsids, envelopes, and nucleic acids are listed below each image. Also, biophysical properties such as size and density are shown, as are peak concentrations of the particles found in blood plasma.

Given their abundance and regulatory functions, it is not surprising that viruses have evolved to usurp EV-mediated signaling. Viruses are obligate intracellular parasites and utilize many of the same cellular pathways as EV to facilitate particle uptake, trafficking, and egress. Several viruses have been shown to incorporate viral nucleic acids and/or proteins into EV. Human herpesviruses such as Epstein-Barr virus (EBV, also known as human herpesvirus 4) and Kaposi’s Sarcoma-Associated Herpesvirus (KSHV, also known as human herpesvirus 8) incorporate viral proteins and miRNAs into EV (Chugh et al. 2013; Dittmer and Damania 2013; Hurwitz et al. 2017, 2018; Meckes et al. 2010, 2013; Yogev et al. 2017; McNamara et al. 2019; Zhao et al. 2019; Pegtel et al. 2010). Human immunodeficiency virus (HIV), as well as its ancestral simian immunodeficiency virus (SIV) incorporate the protein Nef into CD63/CD81+ EV and can deliver it to naïve and uninfectable cells throughout the course of infection (McNamara et al. 2018b; Raymond et al. 2016; Khan et al. 2016; Lenassi et al. 2010; Sami Saribas et al. 2017; Aqil et al. 2014; Mukhamedova et al. 2019; Muratori et al. 2009). In addition to Nef, the HIV-encoded TAR RNA has been detected in EV (Narayanan et al. 2013; Sampey et al. 2016). An even more overt example of virus hijacking the EV biogenesis pathway is incorporation of the full length viral genomic mRNA or the entire viral particle into EV upon infection with hepatitis C virus (HCV) or Hepatitis A virus (HAV), respectively (Feng et al. 2013; McKnight et al. 2017; Rivera-Serrano et al. 2019; Longatti et al. 2015; Bukong et al. 2014; Ramakrishnaiah et al. 2013). Given the propensity of evolutionarily distinct viruses to usurp the EV pathways, it has been proposed the viruses utilize EV as a “Trojan horse” to deliver functional materials for full disease pathogenesis (Gould et al. 2003). Taken together, studies in the field of virology have contributed to our knowledge of EV biogenesis, packaging and delivery, and vice versa.

The isolation of EV or virus particles to high concentration and purity is critical to their study. The separation of these vesicles based on physical parameters alone, such as size, markers, and from contaminants such as protein aggregates has proven to be a non-trivial aspect of their purification. Methods of their purification, as well as distinctive qualities of subclasses of EV have been discussed previously (Théry et al. 2006; Lotvall et al. 2014; Witwer et al. 2013; Reiner et al. 2017; Peterson et al. 2015; Théry et al. 2018; Théry et al. 2002; Stoorvogel et al. 2002). The most prominent methods applicable to both EV and virus isolation include, but are not limited to: (I) ultracentrifugation, (II) precipitation with crowding reagents, (III) crossflow filtration (IV), column chromatography and high performance liquid chromatography (HPLC), (V) immunoprecipitation/affinity capture, and (VI) nanoscale flow cytometry. Additionally, assays that can quantitate the phenotype of cells exposed to EV from sources such as an infected cell or a tumor representa developing field (Raab-Traub and Dittmer 2017; M. Anderson et al. 2018). In this review, we will compare purification strategies of viruses and EV, and recent efforts to characterize the functions they play in cellular homeostasis. Of note, this review is focused on manufacturing and lab-based assays; the emerging field of EV in diagnostics is not covered.

Ultracentrifugation

Given that viruses and EV are low-density particles, only high-speed centrifugation can pellet them directly from solution. For decades, ultracentrifugation (>100,000 * g) has been used to isolate eukaryotic viruses such as influenza, and bacteriophages (Kutner et al. 2009; Reimer et al. 1966, 1967; Sugita et al. 2011; Bachrach and Friedmann 1971). Since virus particles and EV are similar in size and density (Table 1), ultracentrifugation became a frequently employed technique for EV isolation. Given that ultracentrifugation is widely used in routine molecular biology and biochemistry, it presents itself as a rapid and reliable isolation technique for virus and EV isolation. Viruses and EV isolated through ultracentrifugation retain functional capacities such as infectivity and esterase enzymatic activity, respectively. A lingering question, and target for optimization, is the exact amount of biological activity that is maintained throughout purification and which assays are used to define “biological activity” vis-à-vis non-purified biological fluids.

Table 1 Macromolecules such as viruses and extracellular vesicle subclasses are listed, along with their approximate densities and diameters. * For Hepatitis A Virus, the non-enveloped and cell-derived envelope data are merged (see (Feng et al. 2013; McKnight et al. 2017).

The majority of ultracentrifugation instruments are meant for smaller volume inputs (~20–50 mL), but there are some that can hold larger volumes. For this reason, ultracentrifugation is frequently employed as a quick method for the isolation of particles like lentiviruses and EV from these volumes (though exceptions exist, further reviewed in (Li et al. 2017)). Many protocols include creating sucrose or iodixanol gradients to separate the heavy viruses and EV away from protein aggregates, nucleic acid bodies, low-density lipoproteins (LDLs), etc. (Willms et al. 2016, 2018; Iwai et al. 2016; Onódi et al. 2018; Lemon et al. 1985). Repeated ultracentrifugation steps have been shown to decrease EV product yields (Lobb et al. 2015; McNamara et al. 2018a), perhaps due to the tremendous g-force exerted on the vesicles.

In terms of gradients, there are two principles: (I) density gradients used as a cushion, and (II) floatation designs. When used as a cushion, an EV prep is placed at the top of a density gradient (such as iodixanol or sucrose), and ultracentrifugation allows for the sedimentation of macromolecules based on their density. A detailed protocol regarding this approach was published by Théry’s group (Théry et al. 2006). In a floatation design, the mixture is placed at the bottom of a gradient tube, and molecules “float” up to their densities. Denser particles, such as nucleic acid and protein aggregates collect at the bottom, while smaller non-EV associated proteins collect above the EV band. This has allowed researchers to not only separate EV from contaminates, but identify subpopulations within the EV mixture (Bobrie et al. 2012; Jeppesen et al. 2014; Willms et al. 2018, 2016). In herpesvirology, gradient centrifugation has matured to the point were distinct packaging intermediates (A, B and C capsids) can be differentiated and isolated in high enough concentrations to perform biochemical assays and structural analysis (Trus et al. 2001; Deng et al. 2008)

While ultracentrifugation is a durable and time-tested method for the isolation of viruses and EV, there are some caveats to its employment. Isolation using ultracentrifugation results in abnormal size distribution profiles and flattening/lysing of EV (McNamara et al. 2018a; Sugita et al. 2011). Without carefully prepared cushions, viruses and EV will co-sediment with similarly weighted macromolecules such as protein aggregates and LDLs after prolonged spin cycles (Cvjetkovic et al. 2014). Of importance to our discussion, some viruses exhibit overlapping densities with EV such as exosomes and microvesicles, demonstrating that even carefully prepared sucrose/iodixanol/cesium chloride gradients cannot be used to conclusively separate them (Table 1) (Chu and Ng 2004; Feng et al. 2013; Lemon et al. 1985; McKnight et al. 2017; Ivanova et al. 2017; Ettelaie et al. 2014). Therefore, while gradients can be prepared to separate some more dense viruses from EV, co-contamination is expected for lower density families of viruses such as the Flaviviruses.

To conclude, ultracentrifugation is an efficient tool to concentrate viruses and EV from fluids such as tissue culture supernatant for analytical and small-scale experimental biochemistry. Typically, the input requires in excess of 5 mL and becomes impractical if more than 50 mL of input need to be processed. The method is not ideally suited for the separation of EV and viruses, particularly from infected cultures and/or body fluids and it is difficult to adopt ultracentrifugation to large-scale production environments or to environments that present biosafety concerns.

Precipitation with Crowding Reagents

Crowding reagents such as polyethylene glycol (PEG) have been used for many years to precipitate macromolecules and complex protein structures, such as viruse particles, out of solution. This precipitation of viruses and EV allows for lower speed centrifugation. Instruments using both larger (3000 mL) and smaller (0.2 mL) input volumes than ultracentrifugation can be used. PEG is the primary active ingredient in various exosome precipitation reagents that are sold commerically.

Similar to ultracentrifugation, precipitation is thoroughly reliable technique for the concentration of viruses out of solution. Successful precipitation protocols for diverse viruses such as herpesviruses, retroviruses, and bacteriophages exist with PEG and other crowding reagents (Adams 1973; Friedmann and Haas 1970; Orlando et al. 2000; Kutner et al. 2009). Given that viruses and smaller EV such as microvesicles and exosomes have similar biophysical properties, PEG precipitation has become a widely adapted procedure for EV isolation from fluids such as tissue culture supernatant and plasma. The introduction of PEG into a solution like plasma or tissue culture supernatant can be thought of as acting like a molecular fishing net: grabbing larger molecules (depending on the molecular weight of the PEG) into a dense cluster while allowing smaller molecules to be left in the solution. This allows for low-speed centrifugation (< 2000 * g) to pellet the virus and/or EV aggregates (Peterson et al. 2015; Hurwitz et al. 2016; Rider et al. 2016; Hurwitz et al. 2017; McNamara et al. 2018b).

A substantial advantage for crowding reagent precipitation is that it allows for large input volumes. While many ultracentrifuge units are limited based on the volumes that ultracentrifuge-compatible tubes can hold, precipitating using crowding reagents can be done at lower speeds, usually allowing for much larger input volumes as most desktop centrifuges can hold larger volumes (100 mL – 1 L) than ultracentrifuge units. Desktop centrifuge units are much cheaper than ultracentrifuges, and many institutions have an abundance/surplus of the former and a deficiency of the latter. From a cost-of-production standpoint, precipitation with crowding reagents enjoys a wide advantage compared to other methods.

Since PEG precipitation is able to pellet vesicular bodies such as viruses and EV, it suffers from the same caveat as ultracentrifugation: the inability to separate them. PEG precipitation is usually a more time-extended isolation method for EV, requiring hours of precipitation prior to any centrifugation. Like all biological molecules, there exist half-lives for viruses (infectability) and EV (enzymatic and endocytic activity). Therefore, a slight loss in biological activity should be expected. Additionally, electron microscopy (EM) mounting techniques often require the elimination of PEG and other crowding reagents prior to visualization. Crowding reagents also precipitate protein aggregates and non-EV associated extracellular nucleic acids. To that end, additional steps to remove the PEG are advisable prior to any functional assays or imaging (McNamara et al. 2018b; Rider et al. 2016; Chugh et al. 2013; McNamara et al. 2019; Soares Martins et al. 2018).

In sum, precipitation with crowding reagents is another highly successful method for the concentration of extracellular vesicular bodies such as viruses and EV. The ability to take EV or viruses out from a large volume of fluid without high-speed and damaging centrifugations presents itself as a great strength. Conversely, precipitations can take many hours. Precipitation with crowding reagents does more to concentrate viruses and EV rather than separate them away from contaminants.

Crossflow Filtration

An emerging technology for the isolation of viruses and EV from solution is crossflow filtration (also referred to as tangential flow filtration). Crossflow filtration has been used for many years for the bulk production of biologically active molecules such antibiotics and antibodies, particularly in the biotechnology sector where adherence to good manufacturing practices (GMP) is paramount (Lebreton et al. 2008). In this method of filtration, solutions are passed tangentially across a membrane instead of head on. Molecules smaller than the weight cut-off filter are trapped inside the membrane and eventually removed into a discard (permeate) chamber. The solution retaining molecules not captured by the membrane return to the crossflow chamber (retentate) and are continuously pumped through the system as the volume decreases. This results in a concentration of filtered solution in the crossflow chamber. In addition, most crossflow filtration units have an equilibration tank to exchange buffers and wash the filtered solution as it is being concentrated (Fig. 2).

Fig. 2
figure2

Diagram of crossflow filtration. a Design of a basic crossflow filtration unit. A retentate tank is loaded with input solution and processed through. Molecules not meeting the molecular weight cutoff are lost to the permeate while molecules meeting the cutoff are returned to the retentate. A separate tank containing equilibration solution can also be pumped into the retentate tank to equilibrate/wash the filtered and concentrated solution. b Zoomed in representation of hollow-fibers used in crossflow filtration.

Multiple independent groups have proposed using crossflow filtration as a first step in virus and EV isolation (Grzenia et al. 2008; McNamara et al. 2018a; Wickramasinghe et al. 2005; Corso et al. 2017; Castro-Mejía et al. 2015; Busatto et al. 2018; Lamparski et al. 2002). This can be accomplished through the use of high molecular weight membranes, which allow for the retention of macromolecules such as viruses and EV while smaller molecules are lost to the permeate. There are several distinct advantages to using crossflow filtration: (I) industrial-scale input volumes can be used as many crossflow filtration units hold liter-level volumes; (II) buffer exchanges and real-time washing during concentration; (III) a lack of potentially damaging high-speed centrifugation steps.

Since crossflow filtration is an emerging field of concentration and purification of viruses and EV, it is likely that studies and innovations in the coming years will improve upon this technique. As it stands, there are a few pitfalls to employing crossflow that need to be addressed. Like the other methods, crossflow filtration does not allow for the separation of viruses and EV. While hollow-fiber cartridges can vary in their pore size (from purification of antibodies to large virus isolation), viruses and EV have too similar a size to be separated by this method. Therefore, regardless of the conditions used, the separation of viruses from EV is not feasible by exclusively using simple crossflow filtration (McNamara et al. 2018a). Another caveat to using crossflow is that the technology was designed for large input volumes. Using crossflow on a small input volume, such as a few mL of human plasma, but across many samples, would require a new/modified instrument design. Therefore, while crossflow-based isolation of EV and viruses could have major upside in the biotechnology sector for mass production under GMP, its use for analytics and biomarker explorations in smaller clinical specimens is limited.

By contrast to ultracentrifugation and crowding reagent precipitation, crossflow filtration is designed to purify molecules away from contaminants while concentrating them. In that regard, it can be thought of as a hybrid of concentration and purification strategies. While it cannot separate viruses from EV due to overlapping sizes and biophysical properties, it can remove non-virus/EV associated molecules such as extracellular Ago-RNA complexes and albumin, increasing the particle:protein ratio. Albumin removal, in particular, has been the subject of detailed studies as albumin represents the major protein compound of plasma (Arroyo et al. 2011; Webber and Clayton 2013; McNamara et al. 2018a; Welton et al. 2015; Busatto et al. 2018; Grzenia et al. 2008; Wickramasinghe et al. 2005). Similar to column chromatography, crossflow does not exert extreme biophysical forces onto the EV.

Column Chromatography

Column chromatography has been employed for the separation of molecules based on their sizes for decades. There are two main types: exclusion chromatography based on size and chromatography based on charge, hydrophobicity, or another affinity measure. There have been a number of modifications to column chromatography for the enhancement of final molecule purity, biological activity, and binding capacity. New columns have been developed recently to improve the isolation of viruses and removal of albumin (Baranyai et al. 2015):

  1. (A)

    Capto Core resin distributed by General Electric, was specifically designed to remove poultry albumin contaminants in the flu vaccine development pipeline (Blom et al. 2014). This column resin was developed to improve upon other flu isolation and purification strategies such as zonal ultracentrifugation. In recent years, Capto Core has been adapted for the purification of EV. Capto Core resin has a very high capacity, and eluted material retains biological activity – whether it is EV or viruses (James et al. 2016; McNamara et al. 2018a; Reiter et al. 2018; Corso et al. 2017).

  2. (B)

    Another recently developed chromatography-based isolation method is the qEV column from Izon. Independent groups have shown that the qEV columns outperform other methods such as ultracentrifugation in removing excess albumin from EV preparations, increasing the particle:protein ratio and removing non-EV associated contaminants. Importantly, several of these observations were in the context of biological fluids, which are rich in non-EV associated contaminants (Stranska et al. 2018; Lobb et al. 2015; Smith and Daniel 2016; Davis et al. 2019).

As exemplified above, column chromatography for the purification of live-virus and active EV is a technology that has seen considerable advancement in the past few years; however, column chromatography still suffers from a few drawbacks. Perhaps the most consequential drawback to using column chromatography is that it limits the input volume size. This is in proportion to bed volume of the beads. For this reason, several groups have proposed using a concentration step prior to use of specialized columns (McNamara et al. 2018a; Corso et al. 2017; McNamara et al. 2019; Davis et al. 2019). In the case of size exclusion chromatography, EV cannot be separated from viruses given their similar sizes.

Column chromatography has been widely used in academic and industrial settings for decades given its reliability. It is used to elute proteins and complexes into fractions based on their biophysical properties. Because of this, column chromatography is viewed more as a purification strategy as opposed to a concentration strategy. While fractions containing EV will likely be more concentrated than the input solution, chromatography is meant to be used more for the separation of contaminants.

Affinity Purification

Affinity purification, which can involve antibody or specific-protein based capture systems such as streptavidin, is a manner to biochemically purify a discreet population from a heterogeneous mixture. Therefore, this method can be used for separation of viruses and EV given the differences in membrane/capsid composition.

Several antibody-based affinity-capture beads are available for the purification of EV. Most of these capture beads take advantage of the tetraspanin proteins present on the surface of EV. Tetraspanins are incorporated onto the EV membrane during trafficking through the endosomal recycling pathway. The tetraspanin-coated EV (most likely exosomes), are biologically active post capture/elution from the beads, and are competent for uptake by recipient cells (McNamara et al. 2018b; Chugh et al. 2013; McNamara et al. 2019). Moreover, affinity beads can be used to analyze biophysical properties of the immobilized EV through microscopy, sequencing, and flow cytometry analyses (Smith and Daniel 2016; Mukherjee et al. 2016; Peterson et al. 2015; Raab-Traub and Dittmer 2017; Théry et al. 2006).

A similar approach can be taken to isolate viruses based on their surface-exposed proteins. Purification of virus particles from solutions such as blood plasma using antibody-coated beads has been extensively used over the years, with a particular emphasis on vaccine development (Davenport et al. 2011; Sellhorn et al. 2009; Ceglarek et al. 2013). The antibody-mediated capture premise is the same as a typical enzyme-linked immunosorbance assay (ELISA), with the exception that ELISAs are meant to quantitate virus particle concentration and not purify a functional product. Moreover, polymers with a natural affinity for virus epitopes have been used to increase binding capacity (Sakudo et al. 2009, 2016; Patramool et al. 2013; Sakudo and Onodera 2012). Similar to EV, immobilized virus particles can be used for flow cytometry analysis. This premise can be expanded upon to analyze for differences/abundance of certain viral particle subspecies, such as HIV-1 particles containing functional trimeric Env spikes (Arakelyan et al. 2013, 2017).

When it comes to affinity selection of EV and/or viruses, the approaches are limited by the binding capacity of the beads and the ability to elute off a functional product. The binding capacity can be attributable to: (I) affinity of the antibody/polymer for its antigen; (II) accessibility of antigens to antibodies/polymers through steric hindrance on the bead; (III) stability of the epitope on the EV or virus surface. In regard to elution, many protocols utilize either proprietary buffers or acidic glycine to remove EV or viruses from the beads (McNamara et al. 2018b; Jørgensen et al. 2013; McNamara et al. 2019; Tauro et al. 2012). Prolonged presence in an acidic solution denatures proteins, causing a loss in virus infectability and/or EV functionality. Therefore, the use of affinity purification can result in a substantial loss of biological integrity and functionality.

Affinity purification remains the Gold Standard for the isolation of a homogenous entity from a heterogeneous input, including the successful separation of virus particles from EV and vice versa. It maximizes specificity at the cost of sensitivity/yield and functional integrity.

NanoFACS and Flow Virometry

Another methodology to separate viruses from EV that has been explored and improved in recent years is a nanoscale flow cytometry approach. Similar to fluorescence assisted cell sorting (FACS), nanoscale flow cytometry and nanoFACS (also called vesicle flow cytometry) are meant to identify and sort EV subpopulations based on a heterogeneous input population (van der Pol et al. 2018; Nolan and Duggan 2018; Lippé 2018). Limitations to this technology are largely based on the inability of small particles (i.e. EV < 200 nm and both enveloped and non-enveloped viruses) to scatter significant amounts of light. The addition of antigen/substrate-specific fluorophores to the EV/virus mixture, coupled with size exclusion chromatography to remove unbound fluorophores, allows for the particles to scatter enough light for specialized cytometers to differentiate above background (Morales-Kastresana et al. 2017; Tang et al. 2017; Musich et al. 2017; Arakelyan et al. 2013; El Bilali et al. 2017; Loret et al. 2012; Tang et al. 2016). A similar approach has been utilized for endogenously labeled viruses, such as fusion of a GFP to HIV-1 gag protein (Bonar and Tilton 2017; Dale et al. 2011; Hübner et al. 2009).

A powerful advantage to nanoFACS is that it can be employed to rapidly characterize heterogeneous input mixtures without the need to concentrate them first. This is owed to the high concentration of EV released during physiological conditions (109–1011 particles/mL) (Mathieu et al. 2019; Thery et al. 2001, 2018; Raab-Traub and Dittmer 2017), or the high concentration of virus particles released during pathogen challenge (up to 109 infectious units/mL depending on the virus) (Bonar and Tilton 2017; Morales-Kastresana et al. 2017; Tang et al. 2016). Moreover, sorting of EV and viruses based on their fluorescence allows for the recovery of a functional product, similar to cell-based FACS, and the sorted fraction has unsurpassed purity and retains biological activity.

A caveat to nanoFACS and flow virometry is that it sorts the small particles into larger volumes of flow-capture solution. These need to be concentrated to visualize by techniques such as immunoblot. Few standards exist to accommodate for the use of different cytometers by different groups; however nanoFACS presents itself as an incredibly powerful technique to separate viruses and EV from contaminating material.

Phenotypic Characterization

In the isolation of EV and viruses it is critical to properly characterize their biological function and activity in addition to ensuring biophysical homogeneity. While viruses exert easily discernable phenotypes upon infection of a cell, the effects of EV on recipient cell physiology are more diverse, subtler, and less understood. EV have been shown to exert a bevy of effects in recipient cells (Kalamvoki et al. 2014; Bridgeman et al. 2015; Kitai et al. 2017; Baglio et al. 2016; Raab-Traub and Dittmer 2017; Chugh et al. 2013; Barclay et al. 2017; Hurwitz et al. 2017, 2018; Meckes et al. 2010, 2013). Therefore, experimental design must be considered carefully, with all necessary controls accounted for.

When it comes to understanding the phenotype of EV on recipient cells, one tool that has become widely used for their study are infections with viruses. A number of evolutionarily distinct viruses have been shown to usurp EV-mediated signaling (M. Anderson et al. 2018; 2016; Raab-Traub and Dittmer 2017). Therefore a virology-based approach to characterize how EV modulates local homeostasis great promise. In order to properly ascribe an observed phenotype as a consequence of EV addition, experimental groups must account for the ability to separate EV from virus particles in their experimental setup. This can be complicated by the incorporation of viral factors into secreted EV from infected cells such as viral proteins (such as EBV LMP1, HIV/SIV Nef) and nucleic acids (such as HCV RNA, EBV/KSHV miRNAs) (Hurwitz et al. 2017, 2018; Meckes et al. 2010, 2013; Verweij et al. 2011; Yogev et al. 2017; Khan et al. 2016; Kirchhoff et al. 2008; Lee et al. 2016; Lenassi et al. 2010; McNamara et al. 2018b; Narayanan et al. 2013; Pereira and daSilva 2016; Sami Saribas et al. 2017; Mukhamedova et al. 2019; Chugh et al. 2013; McNamara et al. 2018a, 2019; Bukong et al. 2014; Longatti et al. 2015; Ramakrishnaiah et al. 2013). In the case of HAV, in which a fully infectious virion can be incorporated into an EV (Feng et al. 2013; Lemon et al. 1985; McKnight et al. 2017; Rivera-Serrano et al. 2019). Multiple experiments need to be coupled to conclusively show that any viral components detected in an EV preparation are not due to the presence of contaminating virus particles. Such assays include, but are not limited to: (I) viral genome and miRNA-specific q(RT)-PCR, (II) high-sensitivity antigen detection assays, (III) virus propagation assays (plaque assays, viral genome reproduction, etc.) post EV treatment.

Several assays have been utilized to show the functional consequence of EV-uptake, particularly in the context of pathogen challenge. Such functional assays include, but are certainly not limited to: gene expression reprogramming (Hurwitz et al. 2017; Barclay et al. 2017; McNamara et al. 2019), cellular adhesion/migration (Chugh et al. 2013; Koumangoye et al. 2011), signaling pathway activation (Chugh et al. 2013; Meckes et al. 2010; Kalamvoki et al. 2014), cell proliferation (Keller et al. 2009; Koumangoye et al. 2011; Ahsan et al. 2016), cell differentiation (Chowdhury et al. 2015; Webber et al. 2015), and apoptosis (Lenassi et al. 2010; Ren et al. 2011) (see also reviews by (Anderson et al. 2016; 2018; Raab-Traub and Dittmer 2017). Mechanisms by which EV elicit these phenotypes are still being deduced but are likely directly tied to the modified cargo (Fig. 3).

Fig. 3
figure3

Representative image of a phenotypic characterization of EV treatment. Purified EV are added to cells and any number of phenotypic consequences can be monitored. In this case, EV taken from healthy donor (EV) or from KSHV-infected cells (KSHV-EV) were added to cells and Ki67 staining was monitored. An increase in Ki67 positive cells was observed in the KSHV-EV group (also see (McNamara et al. 2019)).

It is important to differentiate functional or phenotypic characterization and biochemical characterization. Characterizations of EV and viruses by approaches such as quantitative polymerase chain reaction (qPCR), nanoparticle tracking analyses (NTA), and protein-specific immunoblotting are not usually phenotypic characterizations. While biochemical assays such as these are critical for proper characterizations of the input EV/viral pool (Théry et al. 2018), the consequence of adding them to cells is of great importance as these fields continue to progress.

Future Directions and Concluding Remarks

The purification of EV and virus particles is a rapidly evolving field. This review was meant to introduce and comment on commonly employed techniques, as well as newly developed technologies or modifications to existing ones (Table 2). This includes the emerging field of nanoFACS and flow virometry. Additionally, this review was composed to cover the basic science, manufacturing and lab-based assays as related to virus and EV purification and characterization; the diagnostic arena was not our focus, and we would refer the interested reader to other reviews (Rodrigues et al. 2018; Wu et al. 2019; Xu et al. 2018).

Table 2 List of employed techniques for the isolation of EV and viruses, along with two pros and cons of each method.

As previously mentioned, one aspect of the EV field of study that is garnering significant attention are mechanisms by which they exert a phenotypic response from recipient cells. Activation of signaling pathways has been observed in cells treated with EV; however, the discreet mechanisms by which EV exert pathway specific activation are still being deduced. The transfer of small molecule agonists and metabolites, for instance as produced by virally infected cells, through EV has been described (Kalamvoki et al. 2014; Zhao et al. 2016). This further supports the model that EV act to maintain equilibrium in their close environment.

Virus particles and EV contain many similarities such as densities, radii, maturation, and the ability to serve as packaging systems for nucleic acids (Bousse et al. 2013; Chu and Ng 2004; Feng et al. 2013; Lemon et al. 1985; Théry et al. 2018; Willms et al. 2016; Vader et al. 2014; Anderson et al. 2018; 2016). Given these many overlapping properties, it is not surprising that differentiating between the two in fluids such as plasma and tissue culture supernatant has proven challenging. Their functions, however, are quite different. Viruses usurp a number of cellular processes for the sole reason of propagation. The emerging picture of EV is that they serve to transmit information locally, maintaining an equilibrium. Given this role, it is not surprising that many viruses have evolved to manipulate EV cargo.

In conclusion, there exist multiple ways that have been developed to isolate, purify and concentrate viruses, and these can easily be applied to EV. At the same time, it is very difficult to separate EV from viruses by these methods for all the aforementioned reasons (Fig. 4). When dealing with mixed populations of EV and viruses, it is imperative that these two entities are separated to avoid misinterpretations of data. Major advances when it comes to isolation of pure and functional EV are needed and are bound to drive the field forward.

Fig. 4
figure4

Summary of virus/EV isolation strategies. The overviewed methods are summarized here. Based on cited literature, estimates were made to judge each procedure on their ability to concentrate viruses/EV, the relative purity of the product, and the ability to separate viruses and EV. The asterisks by ultracentrifugation is to represent that this encompasses gradients as well (through gradients, some viruses and EV can be separated).

References

  1. Adams A (1973) Concentration of Epstein-Barr virus from cell culture fluids with polyethylene glycol. J Gen Virol 20(3):391–394. https://doi.org/10.1099/0022-1317-20-3-391

    CAS  PubMed  Article  Google Scholar 

  2. Ahsan NA, Sampey GC, Lepene B, Akpamagbo Y, Barclay RA, Iordanskiy S, Hakami RM, Kashanchi F (2016) Presence of viral RNA and proteins in exosomes from cellular clones resistant to Rift Valley fever virus infection. Front Microbiol 7:139. https://doi.org/10.3389/fmicb.2016.00139

    PubMed  PubMed Central  Article  Google Scholar 

  3. Anderson MR, Kashanchi F, Jacobson S (2016) Exosomes in viral disease. Neurotherapeutics 13(3):535–546. https://doi.org/10.1007/s13311-016-0450-6

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Anderson M, Kashanchi F, Jacobson S (2018) Role of exosomes in human retroviral mediated disorders. J NeuroImmune Pharmacol 13(3):279–291. https://doi.org/10.1007/s11481-018-9784-7

    PubMed  Article  Google Scholar 

  5. Aqil M, Naqvi AR, Mallik S, Bandyopadhyay S, Maulik U, Jameel S (2014) The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells. J Extracell Vesicles 3. https://doi.org/10.3402/jev.v3.23129

  6. Arakelyan A, Fitzgerald W, Margolis L, Grivel JC (2013) Nanoparticle-based flow virometry for the analysis of individual virions. J Clin Invest 123(9):3716–3727. https://doi.org/10.1172/JCI67042

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Arakelyan A, Fitzgerald W, King DF, Rogers P, Cheeseman HM, Grivel JC, Shattock RJ, Margolis L (2017) Flow virometry analysis of envelope glycoprotein conformations on individual HIV virions. Sci Rep 7(1):948. https://doi.org/10.1038/s41598-017-00935-w

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, Tait JF, Tewari M (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 108(12):5003–5008. https://doi.org/10.1073/pnas.1019055108

    PubMed  PubMed Central  Article  Google Scholar 

  9. Bachrach U, Friedmann A (1971) Practical procedures for the purification of bacterial viruses. Appl Microbiol 22(4):706–715

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Baglio SR, van Eijndhoven MA, Koppers-Lalic D, Berenguer J, Lougheed SM, Gibbs S et al (2016) Sensing of latent EBV infection through exosomal transfer of 5'pppRNA. Proc Natl Acad Sci U S A 113(5):E587–E596. https://doi.org/10.1073/pnas.1518130113

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Baranyai T, Herczeg K, Onódi Z, Voszka I, Módos K, Marton N, Nagy G, Mäger I, Wood MJ, el Andaloussi S, Pálinkás Z, Kumar V, Nagy P, Kittel Á, Buzás EI, Ferdinandy P, Giricz Z (2015) Isolation of exosomes from blood plasma: qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PLoS One 10(12):e0145686. https://doi.org/10.1371/journal.pone.0145686

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Barclay RA, Schwab A, DeMarino C, Akpamagbo Y, Lepene B, Kassaye S, Iordanskiy S, Kashanchi F (2017) Exosomes from uninfected cells activate transcription of latent HIV-1. J Biol Chem 292(28):11682–11701. https://doi.org/10.1074/jbc.M117.793521

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Blom H, Åkerblom A, Kon T, Shaker S, van der Pol L, Lundgren M (2014) Efficient chromatographic reduction of ovalbumin for egg-based influenza virus purification. Vaccine 32(30):3721–3724. https://doi.org/10.1016/j.vaccine.2014.04.033

    CAS  PubMed  Article  Google Scholar 

  14. Bobrie A, Colombo M, Krumeich S, Raposo G, Théry C (2012) Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation. J Extracell Vesicles 1. https://doi.org/10.3402/jev.v1i0.18397

  15. Bonar MM, Tilton JC (2017) High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry. Virology 505:80–90. https://doi.org/10.1016/j.virol.2017.02.016

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Bousse T, Shore DA, Goldsmith CS, Hossain MJ, Jang Y, Davis CT, Donis RO, Stevens J (2013) Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles. J Virol Methods 193(2):589–596. https://doi.org/10.1016/j.jviromet.2013.07.026

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Bridgeman A, Maelfait J, Davenne T, Partridge T, Peng Y, Mayer A, Dong T, Kaever V, Borrow P, Rehwinkel J (2015) Viruses transfer the antiviral second messenger cGAMP between cells. Science 349(6253):1228–1232. https://doi.org/10.1126/science.aab3632

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Bukong TN, Momen-Heravi F, Kodys K, Bala S, Szabo G (2014) Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90. PLoS Pathog 10(10):e1004424. https://doi.org/10.1371/journal.ppat.1004424

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Busatto S, Vilanilam G, Ticer T, Lin WL, Dickson DW, Shapiro S et al (2018) Tangential flow filtration for highly efficient concentration of extracellular vesicles from large volumes of fluid. Cells 7(12). https://doi.org/10.3390/cells7120273

  20. Castro-Mejía JL, Muhammed MK, Kot W, Neve H, Franz CM, Hansen LH et al (2015) Optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage communities in the human gut. Microbiome 3:64. https://doi.org/10.1186/s40168-015-0131-4

    PubMed  PubMed Central  Article  Google Scholar 

  21. Ceglarek I, Piotrowicz A, Lecion D, Miernikiewicz P, Owczarek B, Hodyra K, Harhala M, Górski A, Dąbrowska K (2013) A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display. Sci Rep 3:3220. https://doi.org/10.1038/srep03220

    PubMed  PubMed Central  Article  Google Scholar 

  22. Chevillet JR, Kang Q, Ruf IK, Briggs HA, Vojtech LN, Hughes SM, Cheng HH, Arroyo JD, Meredith EK, Gallichotte EN, Pogosova-Agadjanyan EL, Morrissey C, Stirewalt DL, Hladik F, Yu EY, Higano CS, Tewari M (2014) Quantitative and stoichiometric analysis of the microRNA content of exosomes. Proc Natl Acad Sci U S A 111(41):14888–14893. https://doi.org/10.1073/pnas.1408301111

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Chowdhury R, Webber JP, Gurney M, Mason MD, Tabi Z, Clayton A (2015) Cancer exosomes trigger mesenchymal stem cell differentiation into pro-angiogenic and pro-invasive myofibroblasts. Oncotarget 6(2):715–731. https://doi.org/10.18632/oncotarget.2711

    PubMed  Article  Google Scholar 

  24. Chu JJ, Ng ML (2004) Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. J Virol 78(19):10543–10555. https://doi.org/10.1128/JVI.78.19.10543-10555.2004

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Chugh PE, Sin SH, Ozgur S, Henry DH, Menezes P, Griffith J, Eron JJ, Damania B, Dittmer DP (2013) Systemically circulating viral and tumor-derived microRNAs in KSHV-associated malignancies. PLoS Pathog 9(7):e1003484. https://doi.org/10.1371/journal.ppat.1003484

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Corso G, Mäger I, Lee Y, Görgens A, Bultema J, Giebel B, Wood MJA, Nordin JZ, Andaloussi SEL (2017) Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography. Sci Rep 7(1):11561. https://doi.org/10.1038/s41598-017-10646-x

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Crescitelli R, Lässer C, Szabó TG, Kittel A, Eldh M, Dianzani I, Buzás EI, Lötvall J (2013) Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J Extracell Vesicles 2. https://doi.org/10.3402/jev.v2i0.20677

  28. Cvjetkovic A, Lötvall J, Lässer C (2014) The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. J Extracell Vesicles 3. https://doi.org/10.3402/jev.v3.23111

  29. Dale BM, McNerney GP, Hübner W, Huser TR, Chen BK (2011) Tracking and quantitation of fluorescent HIV during cell-to-cell transmission. Methods 53(1):20–26. https://doi.org/10.1016/j.ymeth.2010.06.018

    CAS  PubMed  Article  Google Scholar 

  30. Davenport TM, Friend D, Ellingson K, Xu H, Caldwell Z, Sellhorn G, Kraft Z, Strong RK, Stamatatos L (2011) Binding interactions between soluble HIV envelope glycoproteins and quaternary-structure-specific monoclonal antibodies PG9 and PG16. J Virol 85(14):7095–7107. https://doi.org/10.1128/JVI.00411-11

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Davis CN, Phillips H, Tomes JJ, Swain MT, Wilkinson TJ, Brophy PM, Morphew RM (2019) The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. PLoS Negl Trop Dis 13(2):e0007191. https://doi.org/10.1371/journal.pntd.0007191

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Deng B, O'Connor CM, Kedes DH, Zhou ZH (2008) Cryo-electron tomography of Kaposi's sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation. J Struct Biol 161(3):419–427. https://doi.org/10.1016/j.jsb.2007.10.016

    CAS  PubMed  Article  Google Scholar 

  33. Dittmer DP, Damania B (2013) Kaposi sarcoma associated herpesvirus pathogenesis (KSHV)--an update. Curr Opin Virol 3(3):238–244. https://doi.org/10.1016/j.coviro.2013.05.012

    PubMed  PubMed Central  Article  Google Scholar 

  34. Effio CL, Hubbuch J (2015) Next generation vaccines and vectors: designing downstream processes for recombinant protein-based virus-like particles. Biotechnol J 10(5):715–727. https://doi.org/10.1002/biot.201400392

    CAS  PubMed  Article  Google Scholar 

  35. El Bilali N, Duron J, Gingras D, Lippé R (2017) Quantitative evaluation of protein heterogeneity within herpes simplex virus 1 particles. J Virol 91(10). https://doi.org/10.1128/JVI.00320-17

  36. Ettelaie C, Collier ME, Maraveyas A, Ettelaie R (2014) Characterization of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures. J Extracell Vesicles 3. https://doi.org/10.3402/jev.v3.23592

  37. Feng Z, Hensley L, McKnight KL, Hu F, Madden V, Ping L, Jeong SH, Walker C, Lanford RE, Lemon SM (2013) A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 496(7445):367–371. https://doi.org/10.1038/nature12029

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Friedmann T, Haas M (1970) Rapid concentration and purification of polyoma virus and SV40 with polyethylene glycol. Virology 42(1):248–250

    CAS  PubMed  Article  Google Scholar 

  39. Gould SJ, Booth AM, Hildreth JE (2003) The Trojan exosome hypothesis. Proc Natl Acad Sci U S A 100(19):10592–10597. https://doi.org/10.1073/pnas.1831413100

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Grzenia DL, Carlson JO, Wickramasinghe SR (2008) Tangential flow filtration for virus purification. J Membr Sci 321(2):373–380. https://doi.org/10.1016/j.memsci.2008.05.020

    CAS  Article  Google Scholar 

  41. Hübner W, McNerney GP, Chen P, Dale BM, Gordon RE, Chuang FY et al (2009) Quantitative 3D video microscopy of HIV transfer across T cell virological synapses. Science 323(5922):1743–1747. https://doi.org/10.1126/science.1167525

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Hurwitz SN, Rider MA, Bundy JL, Liu X, Singh RK, Meckes DG Jr (2016) Proteomic profiling of NCI-60 extracellular vesicles uncovers common protein cargo and cancer type-specific biomarkers. Oncotarget 7(52):86999–87015. https://doi.org/10.18632/oncotarget.13569

    PubMed  PubMed Central  Article  Google Scholar 

  43. Hurwitz SN, Nkosi D, Conlon MM, York SB, Liu X, Tremblay DC, Meckes DG Jr (2017) CD63 regulates Epstein-Barr virus LMP1 Exosomal packaging, enhancement of vesicle production, and noncanonical NF-κB signaling. J Virol 91(5). https://doi.org/10.1128/JVI.02251-16

  44. Hurwitz SN, Cheerathodi MR, Nkosi D, York SB, Meckes DG Jr (2018) Tetraspanin CD63 bridges Autophagic and endosomal processes to regulate Exosomal secretion and intracellular signaling of Epstein-Barr virus LMP1. J Virol 92(5). https://doi.org/10.1128/JVI.01969-17

  45. Ivanova EA, Myasoedova VA, Melnichenko AA, Grechko AV, Orekhov AN (2017) Small dense Low-density lipoprotein as biomarker for atherosclerotic diseases. Oxidative Med Cell Longev 2017:1273042–1273010. https://doi.org/10.1155/2017/1273042

    CAS  Article  Google Scholar 

  46. Iwai K, Minamisawa T, Suga K, Yajima Y, Shiba K (2016) Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations. J Extracell Vesicles 5:30829. https://doi.org/10.3402/jev.v5.30829

    CAS  PubMed  Article  Google Scholar 

  47. James KT, Cooney B, Agopsowicz K, Trevors MA, Mohamed A, Stoltz D, Hitt M, Shmulevitz M (2016) Novel high-throughput approach for purification of infectious Virions. Sci Rep 6:36826. https://doi.org/10.1038/srep36826

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Ørntoft TF, Howard KA, Ostenfeld MS (2014) Comparative analysis of discrete exosome fractions obtained by differential centrifugation. J Extracell Vesicles 3:25011. https://doi.org/10.3402/jev.v3.25011

    CAS  PubMed  Article  Google Scholar 

  49. Jørgensen M, Bæk R, Pedersen S, Søndergaard EK, Kristensen SR, Varming K (2013) Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping J Extracell Vesicles:2. https://doi.org/10.3402/jev.v2i0.20920

  50. Kalamvoki M, Du T, Roizman B (2014) Cells infected with herpes simplex virus 1 export to uninfected cells exosomes containing STING, viral mRNAs, and microRNAs. Proc Natl Acad Sci U S A 111(46):E4991–E4996. https://doi.org/10.1073/pnas.1419338111

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Keller S, König AK, Marmé F, Runz S, Wolterink S, Koensgen D, Mustea A, Sehouli J, Altevogt P (2009) Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes. Cancer Lett 278(1):73–81. https://doi.org/10.1016/j.canlet.2008.12.028

    CAS  PubMed  Article  Google Scholar 

  52. Khan MB, Lang MJ, Huang MB, Raymond A, Bond VC, Shiramizu B, Powell MD (2016) Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aβ(1-42) secretion in SH-SY5Y neural cells. J Neuro-Oncol 22(2):179–190. https://doi.org/10.1007/s13365-015-0383-6

    CAS  Article  Google Scholar 

  53. Kirchhoff F, Schindler M, Specht A, Arhel N, Muench J (2008) Role of Nef in primate lentiviral immunopathogenesis. Cell Mol Life Sci 65(17):2621–2636. https://doi.org/10.1007/s00018-008-8094-2

    CAS  PubMed  Article  Google Scholar 

  54. Kitai Y, Kawasaki T, Sueyoshi T, Kobiyama K, Ishii KJ, Zou J, Akira S, Matsuda T, Kawai T (2017) DNA-containing exosomes derived from Cancer cells treated with Topotecan activate a STING-dependent pathway and reinforce antitumor immunity. J Immunol 198(4):1649–1659. https://doi.org/10.4049/jimmunol.1601694

    CAS  PubMed  Article  Google Scholar 

  55. Koumangoye RB, Sakwe AM, Goodwin JS, Patel T, Ochieng J (2011) Detachment of breast tumor cells induces rapid secretion of exosomes which subsequently mediate cellular adhesion and spreading. PLoS One 6(9):e24234. https://doi.org/10.1371/journal.pone.0024234

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. Kutner RH, Zhang XY, Reiser J (2009) Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 4(4):495–505. https://doi.org/10.1038/nprot.2009.22

    CAS  PubMed  Article  Google Scholar 

  57. Lamparski HG, Metha-Damani A, Yao JY, Patel S, Hsu DH, Ruegg C, le Pecq JB (2002) Production and characterization of clinical grade exosomes derived from dendritic cells. J Immunol Methods 270(2):211–226. https://doi.org/10.1016/s0022-1759(02)00330-7

    CAS  PubMed  Article  Google Scholar 

  58. Lebreton B, Brown A, van Reis R (2008) Application of high-performance tangential flow filtration (HPTFF) to the purification of a human pharmaceutical antibody fragment expressed in Escherichia coli. Biotechnol Bioeng 100(5):964–974. https://doi.org/10.1002/bit.21842

    CAS  PubMed  Article  Google Scholar 

  59. Lee JH, Schierer S, Blume K, Dindorf J, Wittki S, Xiang W, Ostalecki C, Koliha N, Wild S, Schuler G, Fackler OT, Saksela K, Harrer T, Baur AS (2016) HIV-Nef and ADAM17-containing plasma extracellular vesicles induce and correlate with immune pathogenesis in chronic HIV infection. EBioMedicine 6:103–113. https://doi.org/10.1016/j.ebiom.2016.03.004

    PubMed  PubMed Central  Article  Google Scholar 

  60. Lemon SM, Jansen RW, Newbold JE (1985) Infectious hepatitis A virus particles produced in cell culture consist of three distinct types with different buoyant densities in CsCl. J Virol 54(1):78–85

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  61. Lenassi M, Cagney G, Liao M, Vaupotic T, Bartholomeeusen K, Cheng Y et al (2010) HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells. Traffic 11(1):110–122. https://doi.org/10.1111/j.1600-0854.2009.01006.x

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. Li P, Kaslan M, Lee S, Yao J, Gao Z (2017) Progress in exosome isolation techniques (review). Theranostics 7(3):789–804. https://doi.org/10.7150/thno.18133

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. Lippé R (2018) Flow Virometry: a powerful tool to functionally characterize viruses. J Virol 92(3). https://doi.org/10.1128/JVI.01765-17

  64. Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A et al (2015) Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 4:27031. https://doi.org/10.3402/jev.v4.27031

    PubMed  Article  Google Scholar 

  65. Longatti A, Boyd B, Chisari FV (2015) Virion-independent transfer of replication-competent hepatitis C virus RNA between permissive cells. J Virol 89(5):2956–2961. https://doi.org/10.1128/JVI.02721-14

    CAS  PubMed  Article  Google Scholar 

  66. Loret S, El Bilali N, Lippé R (2012) Analysis of herpes simplex virus type I nuclear particles by flow cytometry. Cytometry A 81(11):950–959. https://doi.org/10.1002/cyto.a.22107

    CAS  PubMed  Article  Google Scholar 

  67. Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C et al (2014) Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3:26913. https://doi.org/10.3402/jev.v3.26913

    PubMed  Article  Google Scholar 

  68. Mathieu M, Martin-Jaular L, Lavieu G, Théry C (2019) Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat Cell Biol 21(1):9–17. https://doi.org/10.1038/s41556-018-0250-9

    CAS  PubMed  Article  Google Scholar 

  69. McKnight KL, Xie L, González-López O, Rivera-Serrano EE, Chen X, Lemon SM (2017) Protein composition of the hepatitis A virus quasi-envelope. Proc Natl Acad Sci U S A 114(25):6587–6592. https://doi.org/10.1073/pnas.1619519114

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. McNamara RP, Caro-Vegas CP, Costantini LM, Landis JT, Griffith JD, Damania BA, Dittmer DP (2018a) Large-scale, cross-flow based isolation of highly pure and endocytosis-competent extracellular vesicles. J Extracell Vesicles 7(1):1541396. https://doi.org/10.1080/20013078.2018.1541396

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  71. McNamara RP, Costantini LM, Myers TA, Schouest B, Maness NJ, Griffith JD, Damania BA, MacLean AG, Dittmer DP (2018b) Nef secretion into extracellular vesicles or exosomes is conserved across human and simian immunodeficiency viruses. MBio 9(1). https://doi.org/10.1128/mBio.02344-17

  72. McNamara RP, Chugh PE, Bailey A, Costantini LM, Ma Z, Bigi R, Cheves A, Eason AB, Landis JT, Host KM, Xiong J, Griffith JD, Damania B, Dittmer DP (2019) Extracellular vesicles from Kaposi sarcoma-associated herpesvirus lymphoma induce long-term endothelial cell reprogramming. PLoS Pathog 15(2):e1007536. https://doi.org/10.1371/journal.ppat.1007536

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. Meckes DG, Shair KH, Marquitz AR, Kung CP, Edwards RH, Raab-Traub N (2010) Human tumor virus utilizes exosomes for intercellular communication. Proc Natl Acad Sci U S A 107(47):20370–20375. https://doi.org/10.1073/pnas.1014194107

    PubMed  PubMed Central  Article  Google Scholar 

  74. Meckes DG, Gunawardena HP, Dekroon RM, Heaton PR, Edwards RH, Ozgur S, Griffith JD, Damania B, Raab-Traub N (2013) Modulation of B-cell exosome proteins by gamma herpesvirus infection. Proc Natl Acad Sci U S A 110(31):E2925–E2933. https://doi.org/10.1073/pnas.1303906110

    PubMed  PubMed Central  Article  Google Scholar 

  75. Morales-Kastresana A, Telford B, Musich TA, McKinnon K, Clayborne C, Braig Z, Rosner A, Demberg T, Watson DC, Karpova TS, Freeman GJ, DeKruyff RH, Pavlakis GN, Terabe M, Robert-Guroff M, Berzofsky JA, Jones JC (2017) Labeling extracellular vesicles for nanoscale flow cytometry. Sci Rep 7(1):1878. https://doi.org/10.1038/s41598-017-01731-2

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  76. Mukhamedova N, Hoang A, Dragoljevic D, Dubrovsky L, Pushkarsky T, Low H, Ditiatkovski M, Fu Y, Ohkawa R, Meikle PJ, Horvath A, Brichacek B, Miller YI, Murphy A, Bukrinsky M, Sviridov D (2019) Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells. PLoS Pathog 15(7):e1007907. https://doi.org/10.1371/journal.ppat.1007907

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  77. Mukherjee K, Ghoshal B, Ghosh S, Chakrabarty Y, Shwetha S, Das S et al (2016) Reversible HuR-microRNA binding controls extracellular export of miR-122 and augments stress response. EMBO Rep 17(8):1184–1203. https://doi.org/10.15252/embr.201541930

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  78. Muratori C, Cavallin LE, Krätzel K, Tinari A, De Milito A, Fais S et al (2009) Massive secretion by T cells is caused by HIV Nef in infected cells and by Nef transfer to bystander cells. Cell Host Microbe 6(3):218–230. https://doi.org/10.1016/j.chom.2009.06.009

    CAS  PubMed  Article  Google Scholar 

  79. Musich T, Jones JC, Keele BF, Jenkins LMM, Demberg T, Uldrick TS, Yarchoan R, Robert-Guroff M (2017) Flow virometric sorting and analysis of HIV quasispecies from plasma. JCI Insight 2(4):e90626. https://doi.org/10.1172/jci.insight.90626

    PubMed  PubMed Central  Article  Google Scholar 

  80. Narayanan A, Iordanskiy S, Das R, Van Duyne R, Santos S, Jaworski E et al (2013) Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA. J Biol Chem 288(27):20014–20033. https://doi.org/10.1074/jbc.M112.438895

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  81. Nolan JP, Duggan E (2018) Analysis of individual extracellular vesicles by flow cytometry. Methods Mol Biol 1678:79–92. https://doi.org/10.1007/978-1-4939-7346-0_5

    CAS  PubMed  Article  Google Scholar 

  82. Onódi Z, Pelyhe C, Terézia Nagy C, Brenner GB, Almási L, Kittel Á, Manček-Keber M, Ferdinandy P, Buzás EI, Giricz Z (2018) Isolation of high-purity extracellular vesicles by the combination of Iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma. Front Physiol 9:1479. https://doi.org/10.3389/fphys.2018.01479

    PubMed  PubMed Central  Article  Google Scholar 

  83. Orlando SJ, Nabavi M, Gharakhanian E (2000) Rapid small-scale isolation of SV40 virions and SV40 DNA. J Virol Methods 90(2):109–114

    CAS  PubMed  Article  Google Scholar 

  84. Patramool S, Bernard E, Hamel R, Natthanej L, Chazal N, Surasombatpattana P, Ekchariyawat P, Daoust S, Thongrungkiat S, Thomas F, Briant L, Missé D (2013) Isolation of infectious chikungunya virus and dengue virus using anionic polymer-coated magnetic beads. J Virol Methods 193(1):55–61. https://doi.org/10.1016/j.jviromet.2013.04.016

    CAS  PubMed  Article  Google Scholar 

  85. Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MAJ, Hopmans ES, Lindenberg JL, de Gruijl TD, Wurdinger T, Middeldorp JM (2010) Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A 107(14):6328–6333. https://doi.org/10.1073/pnas.0914843107

    PubMed  PubMed Central  Article  Google Scholar 

  86. Pereira EA, daSilva LL (2016) HIV-1 Nef: taking control of protein trafficking. Traffic 17(9):976–996. https://doi.org/10.1111/tra.12412

    CAS  PubMed  Article  Google Scholar 

  87. Peterson MF, Otoc N, Sethi JK, Gupta A, Antes TJ (2015) Integrated systems for exosome investigation. Methods 87:31–45. https://doi.org/10.1016/j.ymeth.2015.04.015

    CAS  PubMed  Article  Google Scholar 

  88. Raab-Traub N, Dittmer DP (2017) Viral effects on the content and function of extracellular vesicles. Nat Rev Microbiol 15(9):559–572. https://doi.org/10.1038/nrmicro.2017.60

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  89. Ramakrishnaiah V, Thumann C, Fofana I, Habersetzer F, Pan Q, de Ruiter PE, Willemsen R, Demmers JAA, Stalin Raj V, Jenster G, Kwekkeboom J, Tilanus HW, Haagmans BL, Baumert TF, van der Laan LJW (2013) Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells. Proc Natl Acad Sci U S A 110(32):13109–13113. https://doi.org/10.1073/pnas.1221899110

    PubMed  PubMed Central  Article  Google Scholar 

  90. Raymond AD, Diaz P, Chevelon S, Agudelo M, Yndart-Arias A, Ding H, Kaushik A, Jayant RD, Nikkhah-Moshaie R, Roy U, Pilakka-Kanthikeel S, Nair MP (2016) Microglia-derived HIV Nef+ exosome impairment of the blood-brain barrier is treatable by nanomedicine-based delivery of Nef peptides. J Neuro-Oncol 22(2):129–139. https://doi.org/10.1007/s13365-015-0397-0

    CAS  Article  Google Scholar 

  91. Reimer CB, Baker RS, Newlin TE, Havens ML (1966) Influenza virus purification with the zonal ultracentrifuge. Science 152(3727):1379–1381

    CAS  PubMed  Article  Google Scholar 

  92. Reimer CB, Baker RS, Van Frank RM, Newlin TE, Cline GB, Anderson NG (1967) Purification of large quantities of influenza virus by density gradient centrifugation. J Virol 1(6):1207–1216

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  93. Reiner AT, Witwer KW, van Balkom BWM, de Beer J, Brodie C, Corteling RL, Gabrielsson S, Gimona M, Ibrahim AG, de Kleijn D, Lai CP, Lötvall J, del Portillo HA, Reischl IG, Riazifar M, Salomon C, Tahara H, Toh WS, Wauben MHM, Yang VK, Yang Y, Yeo RWY, Yin H, Giebel B, Rohde E, Lim SK (2017) Concise review: developing best-practice models for the therapeutic use of extracellular vesicles. Stem Cells Transl Med 6(8):1730–1739. https://doi.org/10.1002/sctm.17-0055

    PubMed  PubMed Central  Article  Google Scholar 

  94. Reiter K, Aguilar PP, Wetter V, Steppert P, Tover A, Jungbauer A (2018) Separation of virus-like particles and extracellular vesicles by flow-through and heparin affinity chromatography. J Chromatogr A 1588:77–84. https://doi.org/10.1016/j.chroma.2018.12.035

    CAS  PubMed  Article  Google Scholar 

  95. Ren Y, Yang J, Xie R, Gao L, Yang Y, Fan H, Qian K (2011) Exosomal-like vesicles with immune-modulatory features are present in human plasma and can induce CD4+ T-cell apoptosis in vitro. Transfusion 51(5):1002–1011. https://doi.org/10.1111/j.1537-2995.2010.02909.x

    CAS  PubMed  Article  Google Scholar 

  96. Rider MA, Hurwitz SN, Meckes DG Jr (2016) ExtraPEG: A polyethylene glycol-based method for enrichment of extracellular vesicles. Sci Rep 6:23978. https://doi.org/10.1038/srep23978

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  97. Rivera-Serrano EE, González-López O, Das A, Lemon SM (2019) Cellular entry and uncoating of naked and quasi-enveloped human hepatoviruses. Elife 8. https://doi.org/10.7554/eLife.43983

  98. Rodrigues M, Fan J, Lyon C, Wan M, Hu Y (2018) Role of extracellular vesicles in viral and bacterial infections: pathogenesis, diagnostics, and therapeutics. Theranostics 8(10):2709–2721. https://doi.org/10.7150/thno.20576

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  99. Sakudo A, Onodera T (2012) Virus capture using anionic polymer-coated magnetic beads (review). Int J Mol Med 30(1):3–7. https://doi.org/10.3892/ijmm.2012.962

    CAS  PubMed  Article  Google Scholar 

  100. Sakudo A, Baba K, Tsukamoto M, Sugimoto A, Okada T, Kobayashi T, Kawashita N, Takagi T, Ikuta K (2009) Anionic polymer, poly(methyl vinyl ether-maleic anhydride)-coated beads-based capture of human influenza A and B virus. Bioorg Med Chem 17(2):752–757. https://doi.org/10.1016/j.bmc.2008.11.046

    CAS  PubMed  Article  Google Scholar 

  101. Sakudo A, Baba K, Ikuta K (2016) Capturing and concentrating adenovirus using magnetic anionic nanobeads. Int J Nanomedicine 11:1847–1857. https://doi.org/10.2147/IJN.S104926

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  102. Sami Saribas A, Cicalese S, Ahooyi TM, Khalili K, Amini S, Sariyer IK (2017) HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity. Cell Death Dis 8(1):e2542. https://doi.org/10.1038/cddis.2016.467

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  103. Sampey GC, Saifuddin M, Schwab A, Barclay R, Punya S, Chung MC, Hakami RM, Asad Zadeh M, Lepene B, Klase ZA, el-Hage N, Young M, Iordanskiy S, Kashanchi F (2016) Exosomes from HIV-1-infected cells stimulate production of pro-inflammatory cytokines through trans-activating response (TAR) RNA. J Biol Chem 291(3):1251–1266. https://doi.org/10.1074/jbc.M115.662171

    CAS  PubMed  Article  Google Scholar 

  104. Sellhorn G, Caldwell Z, Mineart C, Stamatatos L (2009) Improving the expression of recombinant soluble HIV envelope glycoproteins using pseudo-stable transient transfection. Vaccine 28(2):430–436. https://doi.org/10.1016/j.vaccine.2009.10.028

    CAS  PubMed  Article  Google Scholar 

  105. Smith JA, Daniel R (2016) Human vaginal fluid contains exosomes that have an inhibitory effect on an early step of the HIV-1 life cycle. AIDS 30(17):2611–2616. https://doi.org/10.1097/QAD.0000000000001236

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  106. Soares Martins T, Catita J, Martins Rosa I, da Cruz AB, Silva EO, Henriques AG (2018) Exosome isolation from distinct biofluids using precipitation and column-based approaches. PLoS One 13(6):e0198820. https://doi.org/10.1371/journal.pone.0198820

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  107. Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G (2002) The biogenesis and functions of exosomes. Traffic 3(5):321–330. https://doi.org/10.1034/j.1600-0854.2002.30502.x

    CAS  PubMed  Article  Google Scholar 

  108. Stranska R, Gysbrechts L, Wouters J, Vermeersch P, Bloch K, Dierickx D, Andrei G, Snoeck R (2018) Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma. J Transl Med 16(1):1. https://doi.org/10.1186/s12967-017-1374-6

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  109. Sugita Y, Noda T, Sagara H, Kawaoka Y (2011) Ultracentrifugation deforms unfixed influenza A virions. J Gen Virol 92(Pt 11):2485–2493. https://doi.org/10.1099/vir.0.036715-0

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  110. Tang VA, Renner TM, Varette O, Le Boeuf F, Wang J, Diallo JS et al (2016) Single-particle characterization of oncolytic vaccinia virus by flow virometry. Vaccine 34(42):5082–5089. https://doi.org/10.1016/j.vaccine.2016.08.074

    PubMed  Article  Google Scholar 

  111. Tang VA, Renner TM, Fritzsche AK, Burger D, Langlois MA (2017) Single-particle discrimination of retroviruses from extracellular vesicles by nanoscale flow cytometry. Sci Rep 7(1):17769. https://doi.org/10.1038/s41598-017-18227-8

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  112. Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56(2):293–304. https://doi.org/10.1016/j.ymeth.2012.01.002

    CAS  PubMed  Article  Google Scholar 

  113. Thery C, Boussac M, Veron P, Ricciardi-Castagnoli P, Raposo G, Garin J et al (2001) Proteomic analysis of dendritic cell-derived exosomes: A secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 166(12):7309–7318

    CAS  PubMed  Article  Google Scholar 

  114. Théry C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2(8):569–579. https://doi.org/10.1038/nri855

    CAS  PubMed  Article  Google Scholar 

  115. Théry C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol, chapter 3, unit 3.22. https://doi.org/10.1002/0471143030.cb0322s30

  116. Théry, C., Witwer, K. W., Aikawa, E., Alcaraz, M. J., Anderson, J. D., Andriantsitohaina, R., Antoniou A., Arab T., Archer F., Atkin-Smith G. K., Ayre D. C., Bach J.M., Bachurski D., Baharvand H., Balaj L., Baldacchino S., Bauer N. N., Baxter A. A., Bebawy M., Beckham C., Bedina Zavec A., Benmoussa A., Berardi A. C., Bergese P., Bielska E., Blenkiron C., Bobis-Wozowicz S., Boilard E., Boireau W., Bongiovanni A., Borràs F. E., Bosch S., Boulanger C. M., Breakefield X., Breglio A. M., Brennan M. Á., Brigstock D. R., Brisson A., Broekman M. L.D., Bromberg J. F., Bryl-Górecka P., Buch S., Buck A. H., Burger D., Busatto S., Buschmann D., Bussolati B., Buzás E. I., Byrd J. B., Camussi G., Carter D. R.F., Caruso S., Chamley L. W., Chang Y.T., Chen C., Chen S., Cheng L., Chin A. R., Clayton A., Clerici S. P., Cocks A., Cocucci E., Coffey R. J., Cordeiro-da-Silva A., Couch Y., Coumans F. A.W., Coyle B., Crescitelli R., Criado M. F., D’Souza-Schorey C., Das S., Datta Chaudhuri A., de Candia P., de Santana Jr E. F., de Wever O., del Portillo H. A., Demaret T., Deville S., Devitt A., Dhondt B., di Vizio D., Dieterich L. C., Dolo V., Dominguez Rubio A. P., Dominici M., Dourado M. R., Driedonks T. A.P., Duarte F. V., Duncan H. M., Eichenberger R. M., Ekström K., el Andaloussi S., Elie-Caille C., Erdbrügger U., Falcón-Pérez J. M., Fatima F., Fish J. E., Flores-Bellver M., Försönits A., Frelet-Barrand A., Fricke F., Fuhrmann G., Gabrielsson S., Gámez-Valero A., Gardiner C., Gärtner K., Gaudin R., Gho Y. S., Giebel B., Gilbert C., Gimona M., Giusti I., Goberdhan D. C.I., Görgens A., Gorski S. M., Greening D. W., Gross J. C., Gualerzi A., Gupta G. N., Gustafson D., Handberg A., Haraszti R. A., Harrison P., Hegyesi H., Hendrix A., Hill A. F., Hochberg F. H., Hoffmann K. F., Holder B., Holthofer H., Hosseinkhani B., Hu G., Huang Y., Huber V., Hunt S., Ibrahim A. G.E., Ikezu T., Inal J. M., Isin M., Ivanova A., Jackson H. K., Jacobsen S., Jay S. M., Jayachandran M., Jenster G., Jiang L., Johnson S. M., Jones J. C., Jong A., Jovanovic-Talisman T., Jung S., Kalluri R., Kano S.I., Kaur S., Kawamura Y., Keller E. T., Khamari D., Khomyakova E., Khvorova A., Kierulf P., Kim K. P., Kislinger T., Klingeborn M., Klinke II D. J., Kornek M., Kosanović M. M., Kovács Á. F., Krämer-Albers E.M., Krasemann S., Krause M., Kurochkin I. V., Kusuma G. D., Kuypers S., Laitinen S., Langevin S. M., Languino L. R., Lannigan J., Lässer C., Laurent L. C., Lavieu G., Lázaro-Ibáñez E., le Lay S., Lee M.S., Lee Y. X. F., Lemos D. S., Lenassi M., Leszczynska A., Li I. T.S., Liao K., Libregts S. F., Ligeti E., Lim R., Lim S. K., Linē A., Linnemannstöns K., Llorente A., Lombard C. A., Lorenowicz M. J., Lörincz Á. M., Lötvall J., Lovett J., Lowry M. C., Loyer X., Lu Q., Lukomska B., Lunavat T. R., Maas S. L.N., Malhi H., Marcilla A., Mariani J., Mariscal J., Martens-Uzunova E. S., Martin-Jaular L., Martinez M. C., Martins V. R., Mathieu M., Mathivanan S., Maugeri M., McGinnis L. K., McVey M. J., Meckes Jr D. G., Meehan K. L., Mertens I., Minciacchi V. R., Möller A., Møller Jørgensen M., Morales-Kastresana A., Morhayim J., Mullier F., Muraca M., Musante L., Mussack V., Muth D. C., Myburgh K. H., Najrana T., Nawaz M., Nazarenko I., Nejsum P., Neri C., Neri T., Nieuwland R., Nimrichter L., Nolan J. P., Nolte-’t Hoen E. N.M., Noren Hooten N., O’Driscoll L., O’Grady T., O’Loghlen A., Ochiya T., Olivier M., Ortiz A., Ortiz L. A., Osteikoetxea X., Østergaard O., Ostrowski M., Park J., Pegtel D. M., Peinado H., Perut F., Pfaffl M. W., Phinney D. G., Pieters B. C.H., Pink R. C., Pisetsky D. S., Pogge von Strandmann E., Polakovicova I., Poon I. K.H., Powell B. H., Prada I., Pulliam L., Quesenberry P., Radeghieri A., Raffai R. L., Raimondo S., Rak J., Ramirez M. I., Raposo G., Rayyan M. S., Regev-Rudzki N., Ricklefs F. L., Robbins P. D., Roberts D. D., Rodrigues S. C., Rohde E., Rome S., Rouschop K. M.A., Rughetti A., Russell A. E., Saá P., Sahoo S., Salas-Huenuleo E., Sánchez C., Saugstad J. A., Saul M. J., Schiffelers R. M., Schneider R., Schøyen T. H., Scott A., Shahaj E., Sharma S., Shatnyeva O., Shekari F., Shelke G. V., Shetty A. K., Shiba K., Siljander P. R.M., Silva A. M., Skowronek A., Snyder II O. L., Soares R. P., Sódar B. W., Soekmadji C., Sotillo J., Stahl P. D., Stoorvogel W., Stott S. L., Strasser E. F., Swift S., Tahara H., Tewari M., Timms K., Tiwari S., Tixeira R., Tkach M., Toh W. S., Tomasini R., Torrecilhas A. C., Tosar J. P., Toxavidis V., Urbanelli L., Vader P., van Balkom B. W.M., van der Grein S. G., van Deun J., van Herwijnen M. J.C., van Keuren-Jensen K., van Niel G., van Royen M. E., van Wijnen A. J., Vasconcelos M. H., Vechetti Jr I. J., Veit T. D., Vella L. J., Velot É., Verweij F. J., Vestad B., Viñas J. L., Visnovitz T., Vukman K. V., Wahlgren J., Watson D. C., Wauben M. H.M., Weaver A., Webber J. P., Weber V., Wehman A. M., Weiss D. J., Welsh J. A., Wendt S., Wheelock A. M., Wiener Z., Witte L., Wolfram J., Xagorari A., Xander P., Xu J., Yan X., Yáñez-Mó M., Yin H., Yuana Y., Zappulli V., Zarubova J., Žėkas V., Zhang J.Y., Zhao Z., Zheng L., Zheutlin A. R., Zickler A. M., Zimmermann P., Zivkovic A. M., Zocco D., Zuba-Surma E. K. (2018). Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles, 7(1), 1535750. https://doi.org/10.1080/20013078.2018.1535750

  117. Trus BL, Heymann JB, Nealon K, Cheng N, Newcomb WW, Brown JC, Kedes DH, Steven AC (2001) Capsid structure of Kaposi's sarcoma-associated herpesvirus, a gammaherpesvirus, compared to those of an alphaherpesvirus, herpes simplex virus type 1, and a betaherpesvirus, cytomegalovirus. J Virol 75(6):2879–2890. https://doi.org/10.1128/JVI.75.6.2879-2890.2001

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  118. Vader P, Breakefield XO, Wood MJ (2014) Extracellular vesicles: emerging targets for cancer therapy. Trends Mol Med 20(7):385–393. https://doi.org/10.1016/j.molmed.2014.03.002

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  119. van der Pol E, de Rond L, Coumans FAW, Gool EL, Böing AN, Sturk A, Nieuwland R, van Leeuwen TG (2018) Absolute sizing and label-free identification of extracellular vesicles by flow cytometry. Nanomedicine 14(3):801–810. https://doi.org/10.1016/j.nano.2017.12.012

    CAS  PubMed  Article  Google Scholar 

  120. Verweij FJ, van Eijndhoven MA, Hopmans ES, Vendrig T, Wurdinger T, Cahir-McFarland E et al (2011) LMP1 association with CD63 in endosomes and secretion via exosomes limits constitutive NF-κB activation. EMBO J 30(11):2115–2129. https://doi.org/10.1038/emboj.2011.123

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  121. Wang Z, Deng Z, Dahmane N, Tsai K, Wang P, Williams DR, Kossenkov AV, Showe LC, Zhang R, Huang Q, Conejo-Garcia JR, Lieberman PM (2015) Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes. Proc Natl Acad Sci U S A 112(46):E6293–E6300. https://doi.org/10.1073/pnas.1505962112

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  122. Webber J, Clayton A (2013) How pure are your vesicles? J Extracell Vesicles 2. https://doi.org/10.3402/jev.v2i0.19861

  123. Webber JP, Spary LK, Sanders AJ, Chowdhury R, Jiang WG, Steadman R, Wymant J, Jones AT, Kynaston H, Mason MD, Tabi Z, Clayton A (2015) Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes. Oncogene 34(3):290–302. https://doi.org/10.1038/onc.2013.560

    CAS  PubMed  Article  Google Scholar 

  124. Welton JL, Webber JP, Botos LA, Jones M, Clayton A (2015) Ready-made chromatography columns for extracellular vesicle isolation from plasma. J Extracell Vesicles 4:27269. https://doi.org/10.3402/jev.v4.27269

    PubMed  Article  Google Scholar 

  125. Wickramasinghe SR, Kalbfuss B, Zimmermann A, Thom V, Reichl U (2005) Tangential flow microfiltration and ultrafiltration for human influenza A virus concentration and purification. Biotechnol Bioeng 92(2):199–208. https://doi.org/10.1002/bit.20599

    CAS  PubMed  Article  Google Scholar 

  126. Willms E, Johansson HJ, Mäger I, Lee Y, Blomberg KE, Sadik M et al (2016) Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci Rep 6:22519. https://doi.org/10.1038/srep22519

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  127. Willms E, Cabañas C, Mäger I, Wood MJA, Vader P (2018) Extracellular vesicle heterogeneity: subpopulations, isolation techniques, and diverse functions in Cancer progression. Front Immunol 9:738. https://doi.org/10.3389/fimmu.2018.00738

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  128. Witwer KW, Buzás EI, Bemis LT, Bora A, Lässer C, Lötvall J, Nolte-‘t Hoen EN, Piper MG, Sivaraman S, Skog J, Théry C, Wauben MH, Hochberg F (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2. https://doi.org/10.3402/jev.v2i0.20360

  129. Wu AY, Ueda K, Lai CP (2019) Proteomic analysis of extracellular vesicles for Cancer diagnostics. Proteomics 19(1–2):e1800162. https://doi.org/10.1002/pmic.201800162

    CAS  PubMed  Article  Google Scholar 

  130. Xu R, Rai A, Chen M, Suwakulsiri W, Greening DW, Simpson RJ (2018) Extracellular vesicles in cancer - implications for future improvements in cancer care. Nat Rev Clin Oncol 15(10):617–638. https://doi.org/10.1038/s41571-018-0036-9

    CAS  PubMed  Article  Google Scholar 

  131. Yogev O, Henderson S, Hayes MJ, Marelli SS, Ofir-Birin Y, Regev-Rudzki N, Herrero J, Enver T (2017) Herpesviruses shape tumour microenvironment through exosomal transfer of viral microRNAs. PLoS Pathog 13(8):e1006524. https://doi.org/10.1371/journal.ppat.1006524

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  132. Zhao H, Yang L, Baddour J, Achreja A, Bernard V, Moss T, Marini JC, Tudawe T, Seviour EG, San Lucas FA, Alvarez H, Gupta S, Maiti SN, Cooper L, Peehl D, Ram PT, Maitra A, Nagrath D (2016) Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism. Elife 5:e10250. https://doi.org/10.7554/eLife.10250

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  133. Zhao M, Nanbo A, Sun L, Lin Z (2019) Extracellular vesicles in Epstein-Barr Virus' life cycle and pathogenesis. Microorganisms 7(2). https://doi.org/10.3390/microorganisms7020048

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Acknowledgements

This work was supported by Public Health Service Grant 1R01DA040394 to Dirk Dittmer and a Translational Fellow Award of the AIDS Malignancy Consortium (AMC) to Ryan McNamara as part of the 5UM1CA121947-10.

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McNamara, R.P., Dittmer, D.P. Modern Techniques for the Isolation of Extracellular Vesicles and Viruses. J Neuroimmune Pharmacol 15, 459–472 (2020). https://doi.org/10.1007/s11481-019-09874-x

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Keywords

  • Extracellular vesicles
  • Viruses
  • Exosomes
  • Microvesicles