Viruses, extracellular vesicles (EVs) and extracellular particles (EPs): a crisis of scientific concepts? DOI:10.13140/RG.2.2.18072.06408, ISSN 2753-8176 (online) - Opinion

Ana Pedro1 1Gwyntwr1386 Healthcare CIC, Regus Chester Business Park, Heronsway, Chester, CH49QR, UK Viruses are defined as submicroscopic infectious agents which can only replicate inside the living cells of an organism. Supposedly, viruses are able to infect animals and plants to microorganisms, including bacteria and archea. Moreover, when not inside the cells they infected, viruses exist in the form of individual particles, named virions. Virions are composed accordingly to literature by genetic material (DNA or RNA which encode the structure of the proteins by which the virus acts); by a protein coat, the capsid, which surrounds and protects the genetic material; and in some cases an outside envelope of lipids (1). Curiously, extracellular vesicles (EVs) are lipid bilayer-delimited particles which are naturally released from almost all types of cells and cannot replicate alone as well. Similarly to virions they carry also carry proteins, nucleic acids and lipids(2). Moreover, extracellular particles (EPs) are small protein-nucleic-acid complexes that are not membrane-bound and their mechanisms of biogenesis are unknown (3). Therefore, what is the difference between viruses, an EV, or an EP? Additionally, viruses can be so small as 20 nm in diameter or can even be larger than some bacteria (4), which both overlaps with the supposed size for EVs, (around 20-30 nm to as large as 10 μm or more, although the vast majority of EVs are smaller than 200 nm) (2) and for EPs (smaller than 50nm) (3). In terms of characterization, both viruses and EVs can be characterized by electron microscopy (EM). However, although this technique is very powerful also presents many technical challenges where procedural errors in sample processing or misinterpretations may result in a variety of different morphologies and artifacts (5). Moreover, there are no specific biomarkers for EVs (2) or EPs (3) and the golden-standard test for viruses, the PCR, is not reliable for diagnosis in virology. PCR test employs primers and probes which are unable to detect a putative virus and may only show a sequence, which can represent recombinant genetic material from different non-viral sources (6). The putative ‘virus sequences’ supposedly found by PCR can also be present not only in different microorganisms but also in different particles, such as EVs (7) and lipoproteins (8), where they could easily have been recombined. If there are no specific biomarkers for viruses, EVs, and EPs, the isolation methods proposed for viruses, EVs, and EPs such as cell culture, centrifugation, asymmetric-flow field-flow fractionation, nanoscale flow cytometry approach) (2-3, 9) cannot be reliable either. In conclusion, if neither viruses nor EVs and EPs are not correctly described and thus any specific biomarkers have been found for them, it is thus impossible to clearly make a distinction between them and thus isolate them. References: 1. Pellett et al. (2014). Basics of Virology. Handb Clin Neurol.; 123: 45–66. 2. Pedro A. (2019). QEIOS. The definition of exosome and extracellular vesicles (EV). https://doi.org/10.32388/392054 3. Malkin and Bratman (2020). Bioactive DNA from extracellular vesicles and particles. Cell Death & Disease, 11, Article number: 584 4. Louten J. (2016). Virus Structure and Classification. Essential Human Virology. : 19–29. 5. Pascucci and Scattini (2021). Imaging extracelluar vesicles by transmission electron microscopy: Coping with technical hurdles and morphological interpretation. Biochim Biophys Acta Gen Subj;1865(4):129648. 6. Borger er al (2020): https://cormandrostenreview.com/report/ 7. https://www.nature.com/articles/s41580-020-0251-y 8. https://pubmed.ncbi.nlm.nih.gov/29388141/ 9. Stelzer-Braid et al (2020). Virus isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for diagnostic and research purposes. Pathology.; 52(7): 760–763.