Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, Old Dominion University researchers 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. This 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. These data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent.
Proposed model showing arthropod-borne flaviviruses transmission to
human host via arthropod exosomes
Tick bite or injection/spit of saliva from infected ticks deposits exosomes containing viral infectious RNA or proteins securely to humans. Exosomes derived from tick cells/saliva infect human skin (keratinocytes) or may directly deposit saliva enriched with exosome containing tick-borne viruses into the blood capillaries/vessels. Exosomes derived from vascular endothelial cells containing flaviviruses infect neighboring endothelial cells leading into infection of the peripheral system. Higher viral loads in peripheral tissues increase viremia in blood and eventually allow entry and replication of these arthropod-borne viruses in brain microvascular endothelial cells that line the BBB. Early production and higher loads of flaviviruses in brain endothelial cells would allow these viruses to cross BBB and infect neurons. Infected neurons produce high numbers of exosomes containing infectious RNA and proteins that fuse with cell membranes of naïve neuronal cells thereby infecting neighboring neurons and leading to spread of infection and severe neuronal loss.
Collectively, these 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.