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Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08 Page 13

resulted in OC43 RNA transfer to the recipient cells. Also, viral RNAs were present in EV fractions. Upon
noting that lymphopenia is associated with severe SARS-CoV-2 infections, they generated HEK transfectants
expressing SARS-CoV-2 spike proteins or multiple viral proteins. EVs from these cells were incubated with
T cells; EVs containing multiple viral proteins reduced cell viability, suggesting an EV-based mechanism for
viral-induced lymphopenia.

Martin Olivier (McGill University, Canada) then presented the role of EVs where a virus infects another
pathogen, in this case, the Leishmania RNA virus and the trypanosome Leishmania. The parasite can be
transmitted through the bite of a sandfly, where it infects macrophages and neutrophils (and macrophage
engulf the neutrophils).The parasite replicates as amastigotes in the macrophage, which can then transmit the
amastigotes to another biting sandfly. Martin’s lab has studied how Leishmania hijack macrophage signaling
and innate immune responses for propagation. These studies identified the pathogen metalloprotease GP63
as a virulence factor, but it was clustered in small entities in the macrophage. This led to the discovery of
GP63 (and other virulence factors) in Leishmania exosomes/EVs, and the exosomes modulated macrophage
signaling, inhibiting antimicrobial defenses and inducing inflammatory cell recruitment at the sites of
infection (during a blood meal). Recently, a Leishmania RNA virus (LRV) was discovered that might be
small enough to fit in Leishmania exosomes, raising the possibility that the virus could be transferred via
exosomes. Indeed, the pathogen exosomes contained viral components that were detected biochemically,
and electron microscopy showed the presence of the whole virus in about 30% of the exosomes. The virus
adds to the skin hyperinflammation upon injection of exosomes, aiding and abetting the infection cycle, but
potentially provides a new therapeutic target.

Hameeda Sultana (Old Dominion University, US) brought up another circumstance where host cell
exosomes facilitate viral transmission. Zika virus (ZIKV) induces cell death in cortical neurons, with the
infections in the differentiated and matured neurons. The infected neurons release exosomes containing
viral RNA and proteins, and the exosomes can re-infect naïve cells. Hameeda’s group sought to understand
the mechanisms behind the enhanced exosome release from infected cells that leads to further viral
transmission. Neutral sphingomyelinase 2 (nSMase2; SMPD3), which is involved in numerous points of the
uptake, in endosomal trafficking, and in vesicle release pathways, was found to be a logical player. In infected
neurons, SMPD3 activity was higher in both the cells and their exosomes; silencing it reduced vesicle
release and viral transmission. Thus, it appears that ZIKV induces SMPD3 activity, which leads to greater
vesiculation, and thus increased passage of the virus.

In a setting where EVs may be used to actually mitigate viral disease, Heather Branscome (George Mason
University) presented data on the use of stem cell EVs in the repair of cellular damage. The scenario involves
HIV infection of neurospheres - a novel achievement in itself - as a model of CNS damage such as HIV-
associated neurocognitive disorders (HAND), and the use of reparative stem cell EVs in healing cell damage.
Stem cell therapies have been touted as a valuable means to treat various diseases, but a more potent activity
may reside in stem cell EVs, which have reduced immunogenicity and a more facile handling and storage.
She described the stem cell sources (mesenchymal stem cells, MSCs, and iPSCs) and described the tangential
flow filtration (TFF) methodology for EV isolation. Stem cell (SC) EV cargo included cytokines (FGF2,
VEGF, IL4) that differed between the two stem cell EV sources. The SC EVs could promote cell migration
and endothelial cell tube formation, and the EVs were taken up by neurospheres with long half-lives (up to
9 days). In HIV-infected neurospheres, SC EVs reduced p53 (Ser-15) phosphorylation, suggesting a return
to the cell cycle, and the EVs also reduced amounts of pro-inflammatory cytokines. Using isolated cell
types (neurons, astrocytes, macrophage) exposed to ionizing radiation, SC EV treatment reduced apoptotic
induction. The results suggest the ability of SC EVs to protect and repair cell damage even in the face of a
brain HIV infection.