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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.

Objective: The maturation process of tick-borne encephalitis virus (TBEV) in the tick RA-257 and porcine PS cells was studied by transmission electron microscopy and the E and NS1 proteins were localized in the infected cells., Methods: The porcine PS and tick RA-257 cell lines were infected with TBEV and examined at different time points post infection under an electron microscope. The E and NS1 proteins were localized with monoclonal antibodies on ultrathin cryosections., Results: The first virus particles and virus-induced vesicles appeared inside hypertrophied and dilated rough endoplasmic reticulum (RER) cisternae in PS cells 15 h p.i. In the course of progressing maturation, the virus particles came up inside the Golgi apparatus and then probably left the cell by the exocytic pathway. Free nucleocapsids did not appear. The observed pattern corresponded to a trans-type maturation. The maximum of the infected PS cell survival was about 50 h p.i. Immunolocalization of some viral proteins (the envelope protein E and the nonstructural protein NS1) revealed the proteins in the cytosol and on the membrane of hypertrophied RER cisternae. On the other hand, the maturation process exhibited different features in the case of the tick RA-257 cells. The nucleocapsids appeared in the cytosol 24 h p.i. and enveloped viral particles were observed in the lumen of vacuoles. Infection of RA-257 cells caused only minor ultrastructural changes and resulted in persistent infection. Immunolocalization of viral proteins in the tick cell line also differed. Proteins E and NS1 were localized in the cytosol and on the vacuolar and plasma membranes., Conclusion: The TBEV maturation pathway in the mammalian host cell line differs from the pathway that the virus undergoes in the tick vector cell line., (Copyright 2006 S. Karger AG, Basel.)

Aedes albopictus cells possess a negative cell surface charge of -12.7 mV with an isoelectrophoretic point (IEP) located between pH 3.0 and 4.0. Infection with Mayaro virus rendered the surface of A. albopictus cells less negative reaching a zeta-potential value of -9.7 mV after 100 h of infection. Concomitantly, the IEP of the infected cells were also altered from 3.0-4.0 to 4.0-5.0. Furthermore, the contact angle measurements clearly showed qualitative alterations in the cell surface of infected cells.