Viral Extracellular Vesicles known as ‘Stealth Spheres’

Pyrrhus

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Given the recent interest in exosomes, I thought I might share some research on extracellular vesicles that the media have, somewhat sensationally, dubbed “Stealth Spheres”:

Chen et al. said:
A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.
Altan-Bonnet said:
Extracellular vesicles have recently emerged as a novel mode of viral propagation exploited by both enveloped and non-enveloped viruses. In particular non-enveloped viruses utilize the hosts' production of extracellular vesicles to exit from cells non-lytically and to hide and manipulate the immune system. Moreover, challenging the long held idea that viruses behave as independent genetic units, extracellular vesicles enable multiple viral particles and genomes to collectively traffic in and out of cells, which can promote genetic cooperativity among viral quasispecies and enhance the fitness of the overall viral population.
Santiana et al. said:
In enteric viral infections, such as those with rotavirus and norovirus, individual viral particles shed in stool are considered the optimal units of fecal-oral transmission. We reveal that rotaviruses and noroviruses are also shed in stool as viral clusters enclosed within vesicles that deliver a high inoculum to the receiving host. Cultured cells non-lytically release rotaviruses and noroviruses inside extracellular vesicles. In addition, stools of infected hosts contain norovirus and rotavirus within vesicles of exosomal or plasma membrane origin. These vesicles remain intact during fecal-oral transmission and thereby transport multiple viral particles collectively to the next host, enhancing both the MOI and disease severity. Vesicle-cloaked viruses are non-negligible populations in stool and have a disproportionately larger contribution to infectivity than free viruses. Our findings indicate that vesicle-cloaked viruses are highly virulent units of fecal-oral transmission and highlight a need for antivirals targeting vesicles and virus clustering.
References:
(Chen et al., 2015) https://www.cell.com/cell/fulltext/...m/retrieve/pii/S0092867415000756?showall=true
(Altan-Bonnet, 2016) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4983493/
(Santiana et al., 2018) https://www.ncbi.nlm.nih.gov/pubmed/30092198
 

Hip

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This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles
I find this interesting from the perspective of viral entry into cells. In the case of coxsackievirus B, this enters cells by the CAR receptor, and so you might expect that cells which lack CAR would be immune to coxsackievirus B infection.

But if enterovirus can spread cell-to-cell by extracellular vesicles, this may allow coxsackievirus B to infect even cells which lack the CAR receptor.

Enterovirus is thought may also spread cell-to-cell by the formation of cellular protrusions, and by intercellular bridges.
 
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I'd like to understand more whether the exhaustive searches for viral DNA could possibly exclude viruses encased inside exosomes. It seems to me likely they would exclude it.

It can't be the case that these viruses that travel by exosome never cause a cell to burst and spread viruses everywhere in the classic viral infection style. But perhaps that is wrong and we could have hidden enteroviruses? Who is our best virus hunter? Who would know? Didn't OMF study this?
 

Sidny

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Who is our best virus hunter? Who would know? Didn't OMF study this?
One of the supposed best “Ian lipkin” is asleep at the wheel imo. These posts below are in regard to enteroviruses which are linked to ME, Parkinson’s etc. but I think the same could be said for other neurotrophic viruses like HSV 1-2, HHV6 etc and its not like any of these studies include biopsies of brain and other relevant nervous system tissue, in living patients anyway.

ME looks a lot like post polio or smoldering/ongoing viral brain injury and this exosome work just further solidifies that notion for me. It seems In line with evidence of noncytolytic enteroviral infections @Hip has done great work on and Dr Martin learners work on abortive herpes virus infections.

“past infection, or agent no longer present” just doesn’t really seem to make much sense to me in regard to ME and other similar illnesses
5A4411E4-A2B0-46CD-B539-D38686AB2F48.png
C0AF53D6-8B5B-41B7-BBA8-7A17770075FB.png
 
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Pyrrhus

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I'd like to understand more whether the exhaustive searches for viral DNA could possibly exclude viruses encased inside exosomes. It seems to me likely they would exclude it.
That is an excellent question. The answer depends upon the specific details of the isolation step of the assay being used. These details are not always fully disclosed in publications, so it is not always possible to know by reading a paper whether or not the author’s work would have looked at the contents of extracellular vesicles.

Who is our best virus hunter?
Unfortunately, the only virologist dedicated to ME/CFS research is Ian Lipkin. Charles Chiu dabbles in ME research from time to time. Ron Davis is not a virologist.

Didn't OMF study this?
No. Ron Davis has never looked at RNA viruses such as the ones described in this thread. Ron Davis has only looked for DNA viruses.
 

Hip

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Unfortunately, the only virologist dedicated to ME/CFS research is Ian Lipkin. Charles Chiu dabbles in ME research from time to time. Ron Davis is not a virologist.
That's true, I don't think there are any other virologists who have a major interest in ME/CFS.

Prof Nora Chapman had a passing interest in ME/CFS after she figured out the mechanism by which enterovirus can persist in the body as a chronic low-level non-cytolytic infection, and then attended a few ME/CFS conferences to explain her research on persistent enterovirus to ME/CFS scientists.

Virologist Prof Ralph Feuer would make a great ME/CFS researcher, as his interest is in enterovirus infections of the central nervous system. But so far he has not done any work in ME/CFS.

Prof Ian Lipkin has had a longstanding interest in ME/CFS, but does not believe enterovirus could cause ME/CFS (he told me this when I contacted him by email). He thinks some other pathogen is responsible, which is why he keeps on hunting for it.
 
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I've never previously subscribed to the ongoing infection school of thought. And that means a lot of this chat goes over my head. I need to google "cytolitic" and also the difference between DNA and RNA viruses!
 
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When it comes to ongoing infection vs “hit and run” I am in neither side. What would possibly produce an immune response like sepsis that includes both immune weakening and increased inflammation ? What could cause massive oxidative stress and downregulation of metabolism and many flu like symptoms but not involve a detectable pathogen? Or involve activation of pathogens that are already present in healthy people? I have some beliefs about this but I generally think that the infectious vs autoimmune/hit and run debate is oversimplified. There could be another factor
 
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Just stumbled onto this special edition of the Journal Proteomics focused on exosomes. I'm not sure there's aything specific in the below for me/cfs but it's good to see how much work is happening on them.

One great thing about exosomes is they are relevant to the best-funded disease of all: cancer. So we can expect lots of progress in understanding them.

Part II: Special Issue on Extracellular Vesicles and Exosomes

Richard J. Simpson

David W. Greening

First published: 16 April 2019

https://doi.org/10.1002/pmic.201900121


Part 1 https://onlinelibrary.wiley.com/toc/16159861/2019/19/1-2


Part 2 https://onlinelibrary.wiley.com/toc/16159861/2019/19/8



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The ability of secreted extracellular vesicles (EVs) to target and transfer their cargo from donor to acceptor cells to trigger phenotypic changes in the latter has generated immense interest in the biological and commercial sectors. EVs are critical mediators of intercellular communication and regulate pleiotropic biological processes in target cells through horizontal transfer of specifically loaded protein, DNA, lipid, and RNA species. Importantly, EV cargo composition varies depending on cell type and cell environment stress. Not surprisingly, EVs have immense therapeutic potential and their cargos are receiving much attention as potential biomarkers for disease diagnosis and prognosis. EVs are present in most body fluids and as such are attractive non‐invasive candidates for disease diagnostics as well as longitudinal therapeutic response metrics of diseases such as cancer, heart disease, and neurodegeneration, in addition to rejection metrics for renal allografts.

The field of EVs is a rapidly growing area of research, with the identification of new types of EVs, expansion on the cell types or organisms that release EVs and their functions, sub‐populations of EV types, as well as the development of novel approaches to characterize and understand EVs. Characterization of distinct types of EV cargo is of significant interest due to the information this cargo provides with respect to the biogenesis, trafficking, targeting, uptake, and cellular effects of EVs. EVs can be classified into two broad categories: exosomes (endosomal origin) and microparticles (also referred to as shed microvesicles and ectosomes, amongst others), which are derived from blebbing of the plasma membrane. Because it is difficult to separate EV classes (and their subtypes) one from another by conventional separation, they are loosely termed “small EVs” and “large EVs.”

Reviewed previously (Greening et al., Expert Rev. Proteomics 2017, 14, 69), mass spectrometry–based proteomics is a powerful technology for the quantitative identification of protein components of EV classes and their subtypes—information that is fundamental for understanding their biogenesis, function, as well as discovery of stereospecific protein markers that might allow EV subtype discrimination. We present a collection of research and review reports that explore methodologies for the isolation and characterization of EVs, biological insights, and therapeutic potential of EVs released from placental cells, stem/stromal cells, immune cells, tumor cells, as well as pathogens and fungus. This Special Issue published in two parts (January and April) includes reviews, research articles, and viewpoints on the study and understanding of EVs, raising important elements of how different systems regulate the composition of EV content, and subsequently how EVs reprogram their target cell proteome. This includes, but is not limited to cancer biology and oncogenic transformation (https://doi.org/10.1002/pmic.201800169, https://doi.org/10.1002/pmic.201800148, https://doi.org/10.1002/pmic.201800180) and bacteria and their diversity of outer membrane vesicles (https://doi.org/10.1002/pmic.201800209). Vagner et al. (https://doi.org/10.1002/pmic.201800167) reviewed how the protein composition reflects EV heterogeneity, directly comparing the protein composition of different EV classes and EV populations derived from the same cell source, as well as different cell types, providing important implications in the understanding of EV biology.

Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and provide an attractive source of disease markers, they are poorly understood. Xu et al. (https://doi.org/10.1002/pmic.201800453) employed a combination of carbonate extraction and TX114 phase separation and mild proteolysis (proteinase K) to fractionate peripherally associated and integral membrane exosomal proteins. Surfaceome proteins were identified using label‐free quantitative mass spectrometry and membrane bioinformatics. Interestingly, this study revealed RNA binding proteins/ribonucleoproteins and RNA species to be exposed on the outer membrane surface of exosomes. To understand how EVs regulate target cell function, Rai et al. (https://doi.org/10.1002/pmic.201800148) investigated how exosomes from distinct cancer cell types reprogram the proteome and function of fibroblasts following transfer. Specifically, this study highlights the role of primary and metastatic tumor‐derived exosomes in generating phenotypically and functionally distinct subsets of cancer‐associated fibroblasts that facilitate tumor progression through oncogenic transformation and metabolic reprogramming.

Studies by Hallal et al. (https://doi.org/10.1002/pmic.201800157) investigated the prognostic potential of vesicular protein cargo (specifically key molecular chaperones) from neurosurgical aspirates of glioblastoma using quantitative mass spectrometry–based proteomics. Further, data independent acquisition (SWATH) by Jayabalan et al. (https://doi.org/10.1002/pmic.201800164) was used to understand physiological mechanisms associated with insulin resistance during human gestation, specifically identifying differentially abundant exosome cargo associated with gestational diabetes mellitus in comparison to normal glucose tolerance.

The therapeutic potential of EVs, specifically mesenchymal stem/stromal cell–derived vesicles (MSC‐EVs), with van Balkom et al. (https://doi.org/10.1002/pmic.201800163) identifying a common protein signature that may be useful in ensuring the homogeneity of EV‐specific therapeutics. Further, commentary by Roura et al. (https://doi.org/10.1002/pmic.201800397) discusses the potential focus areas and issues for rational design and optimization of MSC‐EV production and potency for therapeutics, in addition to Ben‐Hur et al. (https://doi.org/10.1002/pmic.201800170), discussing the potential for microbial‐associated vesicle‐mediated gene delivery. Nasiri et al (https://doi.org/10.1002/pmic.201800161). investigated the generation of artificial EVs, termed cell‐derived mimetic nanovesicles (M‐NVs), a promising alternative to EVs for clinical applicability, comparing not only the content of mimetic nanovesicles and how this differed from the parental cell proteome, but further with purified endosomally derived exosomes using a comprehensive characterization and mass spectrometry approach. Further, this study highlights differences in protein post‐translational modifications among M‐NVs, as distinct from exosomes, using a nontargeted informatic approach, specifically showing phosphorylation, ubiquitination, and thiophosphorylation as protein modifications in M‐NVs.

Key reviews of the field include Taylor et al. (https://doi.org/10.1002/pmic.201800165) highlighting how specific proteins regulate the formation of microvesicles and their drug‐sensitivity capacity in cancer, and Wu et al. (https://doi.org/10.1002/pmic.201800162), Ruhen et al. (https://doi.org/10.1002/pmic.201800155), and Choi et al. (https://doi.org/10.1002/pmic.201800169) providing further insights into EVs and cancer diagnostics. Leading research and guidelines to EV proteomics to cardiovascular disease were highlighted by Barrachina et al., (https://doi.org/10.1002/pmic.201800248) and reviewed by Barrachina et al., (https://doi.org/10.1002/pmic.201800247) describing insights into the clinical relevance and potential of novel EV markers identified in the context of cardiovascular disease.

The field of EVs is a rapidly growing area of basic, applied, and biomedical research, with the identification of new types of EVs, their biology and functions, as well as the development of novel approaches to purify, characterize, and understand EVs. This research topic has covered a number of cutting‐edge discoveries in this field—and importantly, how proteomics is advancing key questions in the field. Proteomics now holds the promise of identification, quantification, and validation of EV proteins and determining EV subtype‐specific markers and biological insights in how EVs modulate target cells, directed toward biomedical research and clinical applications. We predict an innovative and bright future for expanding the application of mass spectrometry proteomics to research in the EV biology community. We thank all contributors to this special issue research topic and the referees for their prompt and in‐depth reviews.

Kind regards,this special edition of the Journal Proteomics focused on exosomes:
 
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It doesn't have to be viruses in the exosomes. It could be MicroRNAs, as this paper explains:

Front Physiol. 2014; 5: 39.

Does regulation of skeletal muscle function involve circulating microRNAs?
Wataru Aoi1,* and Kunihiro Sakuma2

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3925823/

They prpose MiRNAs as potential disease biomarkers and signals of fatigue.

What's interesting here is a detail in the paper. They say that exosome creation ties in with the sphingolipids and ceramides that Naviaux found were so awry:

"Although a detailed mechanism for the secretion of miRNAs from cells has not yet been established, Kosaka et al. (2010b) reported that miRNA secretion was regulated by neutral sphingomyelinase 2, a rate limiting enzyme in the biosynthesis of ceramide, which triggers the secretion of exosomes."

Here's the quote from the Naviaux paper: "In males, over 50% (16/30) of the sphingolipids that were decreased were ceramides, and 47% (14/30) were sphingomyelin species. In females, 86% (18/21) were ceramides and 14% (3/21) were sphingomyelins in females."

In 2014 the Griffith university researchers linked MiRNA to ME/CFS: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102783

Now, this is not conclusive proof exosomes are involved. Whenever something new is turned up by basic science, it's natural to try to tie it to ME/CFS. Whether it's psychology, the microbiome, MiRNAs or exosomes, this pattern repeats. You want to match the new mechanisms to the existing mysteries.

There is no guarantee that there really is a link here to me/cfs. It could be a dead end, but an exosome hypothesis is definitely worth testing.
 
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Hypothesis: me/cfs is a failure of lipid metabolism that manifests in energy deficiencies, and also a deficiency of certain cellular membrane structures that rely on those lipids. The membrane deficiencies lead to problems in the gut, the endothelial system, the blood-brain barrier, connective tissues and problems with production of exosomes.
 

junkcrap50

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It doesn't have to be viruses in the exosomes. It could be MicroRNAs, as this paper explains:
miRNAs are good candidates as they are often the cargo in exosomes and other CFS researchers have found unique miRNAs in CFS.

What's interesting here is a detail in the paper. They say that exosome creation ties in with the sphingolipids and ceramides that Naviaux found were so awry:
Yes, I found the same thing and mentioned it in another post of mine. Looks like sphingolipids play a role in exosome release. One exosome release inhibitor I found functions by disrupting sphingolipid formation. It nicely ties into Naviaux's work.
 
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Hypothesis: me/cfs is a failure of lipid metabolism that manifests in energy deficiencies, and also a deficiency of certain cellular membrane structures that rely on those lipids
Hypothesis: I'm really intrigued by Collagen. Here I see collagen is meeting up with the Lipid metabolism. I intend to read thru this: incrementally. And the not such a co-incidence with all the issues going on with brain stem and cervical spine. I see Collagen issues.

This is just the first random article that appeared with both mentioned.

https://www.cambridge.org/core/jour.../3575189416F7861B875ED46B2E5A815E/core-reader

Since I seem to have to heal myself..... I allow intuitive things to arise and think about them. Toss them into the pot. Stir. See what comes out.

Consuming more bone broths is helping alot of my symptoms.
 

Pyrrhus

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A new publication on extracellular vesicles (such as exosomes) in ME:
https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-020-02560-0

Highlights:
  1. ME patients had more extracellular vesicles of size 30-130nm than healthy controls.
  2. The researchers only looked at cytokines in the extracellular vesicles, not at other contents of the extracellular vesicles.
  3. The researchers did not find any difference in concentrations of cytokines in extracellular vesicles when comparing ME patients with healthy controls.
This work raises some questions:
  1. Why do ME patients have more extracellular vesicles of size 30-130nm?
  2. Where do these extracellular vesicles come from?
  3. What exactly is inside these extracellular vesicles?

Excerpt:
Giloteaux et al 2020 said:
Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome: a pilot study

Background
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of unknown etiology lasting for a minimum of 6 months but usually for many years, with features including fatigue, cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Dysregulation of cytokine signaling could give rise to many of these symptoms. Cytokines are present in both plasma and extracellular vesicles, but little investigation of EVs in ME/CFS has been reported. Therefore, we aimed to characterize the content of extracellular vesicles (EVs) isolated from plasma (including circulating cytokine/chemokine profiling) from individuals with ME/CFS and healthy controls.

Methods
We included 35 ME/CFS patients and 35 controls matched for age, sex and BMI. EVs were enriched from plasma by using a polymer-based precipitation method and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and immunoblotting. A 45-plex immunoassay was used to determine cytokine levels in both plasma and isolated EVs from a subset of 19 patients and controls. Linear regression, principal component analysis and inter-cytokine correlations were analyzed.

Results
ME/CFS individuals had significantly higher levels of EVs that ranged from 30 to 130 nm in size as compared to controls, but the mean size for total extracellular vesicles did not differ between groups. The enrichment of typical EV markers CD63, CD81, TSG101 and HSP70 was confirmed by Western blot analysis and the morphology assessed by TEM showed a homogeneous population of vesicles in both groups. Comparison of cytokine concentrations in plasma and isolated EVs of cases and controls yielded no significant differences. Cytokine-cytokine correlations in plasma revealed a significant higher number of interactions in ME/CFS cases along with 13 inverse correlations that were mainly driven by the Interferon gamma-induced protein 10 (IP-10), whereas in the plasma of controls, no inverse relationships were found across any of the cytokines. Network analysis in EVs from controls showed 2.5 times more significant inter-cytokine interactions than in the ME/CFS group, and both groups presented a unique negative association.

Conclusions
Elevated levels of 30-130 nm EVs were found in plasma from ME/CFS patients and inter-cytokine correlations revealed unusual regulatory relationships among cytokines in the ME/CFS group that were different from the control group in both plasma and EVs. These disturbances in cytokine networks are further evidence of immune dysregulation in ME/CFS.
 

Pyrrhus

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I recently found this review paper from the great Karla Kierkegaard:
https://pubmed.ncbi.nlm.nih.gov/25890822/

Among other things, it discusses how enteroviruses encase themselves inside intracellular vesicles which then exit the cell as extracellular vesicles.

You can see pictures of these extracellular vesicles in A, C, D, and E of this figure:
F7C00792-C528-497B-AC5E-FC16E15626E9.jpeg


They also provide a little background to the study of non-lytic infections by enteroviruses:
52E87924-5974-4BEB-8142-5DF7FE405AF7.jpeg
 

Pyrrhus

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For those of you wondering what the difference is between an "exosome" and an "extracellular vesicle":
  • A "vesicle" is like a bubble. It is surrounded by a fatty membrane and is filled with water and other things.
  • An "extracellular vesicle" is any vesicle that is found outside a cell.
  • An "exosome" is a special type of extracellular vesicle derived from an "endosome".

A new publication on extracellular vesicles (such as exosomes) in ME:
https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-020-02560-0

Highlights:
  1. ME patients had more extracellular vesicles of size 30-130nm than healthy controls.
Since exosomes are typically of size 30-150nm, the extracellular vesicles in this publication could well be exosomes.

I recently found this review paper from the great Karla Kierkegaard:
https://pubmed.ncbi.nlm.nih.gov/25890822/

Among other things, it discusses how enteroviruses encase themselves inside intracellular vesicles which then exit the cell as extracellular vesicles.

You can see pictures of these extracellular vesicles in A, C, D, and E of this figure
These extracellular vesicles containing enteroviruses are not exosomes, and appear to have sizes roughly from 40-800nm.
 
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