Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

Abstract

Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.

Assessing diagnostic value of microRNAs from peripheral blood mononuclear cells and extracellular vesicles in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

“…3). This vesicle subset mediating long-range communication, reportedly carrying differential miRNA cargos in disease [30][31][32][33] , have been shown to have potential diagnostic value in other contexts and, therefore, were of interest to our project. However, as our final goal was to find a technically simple diagnostic strategy with independence of the isolated EV subsets by our method of choice, no further efforts in exosomal marker detection were invested, nor additional purification steps were performed.…”

Wonders of tick saliva

“…2) . This approach may be particularly effective if exosomes secreted in tick saliva play an important role in tick-borne virus transmission (Zhou et al, 2018).…”

Exosomes: Biological Couriers with Transformative Messages

“…This manipulative mechanism has been reported for HIV [34], EBV [35], HCV [36], as well as bacterial pathogens, specifically Mycobacterium tuberculosis [28] [37]. Recently, a similar mechanism has been uncovered for exosomal-mediated transmission of flavivirus RNAs and proteins to humans from arthropods such as the Ixodes scapularis 7 ticks [38]. The Langat virus (LGTV) is suggested to hijack the host tick exosomal system; the transformed exosomes -now particles of high infectivity -delivers the viral cargo to human cells.…”