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.

Wenshuo Zhou,Michael B Sherman,Biswas Neupane,Hameeda Sultana,Michael Woodson,Kyung H Choi,Fengwei Bai,Girish Neelakanta,Aleem Siddiqui

doi:10.1371/journal.ppat.1006764

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.

Highlights

Exosomes are small membranous extracellular microvesicles (30 to 250 nm in diameter) of endocytic origin formed in late endosomal compartments of several different cell types [1,2,3,4,5]
In this study we have demonstrated that cells from the medically important vector tick, secretes exosomes that mediate transmission of tick-borne Langat (LGTV) viruses from arthropod to human and other vertebrate host cells
We have demonstrated that cells from the medically important vector tick, Ixodes scapularis, secretes exosomes that mediate transmission of tickborne Langat virus (LGTV) RNA and proteins from arthropod to human

Summary

Introduction

Exosomes are small membranous extracellular microvesicles (30 to 250 nm in diameter) of endocytic origin formed in late endosomal compartments (as multivesicular bodies; MVBs) of several different cell types [1,2,3,4,5]. Exosomes were considered as garbage bins to discard the unwanted cellular or molecular components or membranous proteins from reticulocytes [6,7,8,9]. Exosomes derived from several different cells have been shown to function as signaling related vesicles, transporting cell-specific collections of several proteins, lipids and nucleic acids such as DNA, RNA and microRNA [12,20,21,22,23,24,25,26,27,28]. Cell-derived exosomes have been shown to be important modes of intercellular communication and as transmitters of information over longer distances for e.g., between different tissues or multiple organs [2,15,27,30,31]

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Results

Conclusion