# Insect-borne non-enveloped bluetongue virus utilizes discrete small vesicles for non-lytic release and cell-to-cell transmission

## TL;DR

Bluetongue virus uses small vesicles to spread between cells without killing them, and these vesicles are rich in a viral protein that aids transmission.

## Contribution

The study identifies two distinct extracellular vesicle types used by BTV for non-lytic release and highlights the role of the NS3 protein in small vesicle formation.

## Key findings

- Bluetongue virus uses both large and small extracellular vesicles for non-lytic release.
- Small extracellular vesicles are enriched with the viral NS3 protein and are more infectious.
- Non-lytic release via vesicles promotes efficient cell-to-cell transmission of the virus.

## Abstract

Bluetongue virus (BTV) is one of the most economically relevant orbiviruses and is the only example of a large complex, but non-enveloped arbovirus. In addition to cell lysis, BTV is known to employ a ‘budding’ process analogous to that used by enveloped viruses for cell exit, in which the viral glycosylated NS3 protein plays a key role. Recent reports have demonstrated that BTV can also induce non-lytic release via extracellular vesicles (EVs), however, details of the type and origin of the EV used and the role of NS3 in the process remain incompletely understood. In this study we undertook biochemical studies on the non-lytic release of BTV particles in different forms of EVs from several types of host cells and complemented this by comprehensive microscopic analyses using fluorescence microscopy, transmission electron microscopy and electron cryo-tomography. We discovered that BTV particles use both large EVs (LEVs) and smaller size EVs (SEVs) for non-lytic release and that, in each cell type studied, SEV fractions were particularly enriched with NS3. Non-enveloped BTV particles initially released in SEVs were highly infectious and promote efficient cell-to-cell transmission. This discovery highlights the complex mechanisms utilized by a non-enveloped arbovirus for egress and the significance of different EV types in this process.

BTV is an emerging arthropod-borne non-enveloped virus causing epidemics worldwide. The recent discovery that BTV and other non-enveloped viruses are secreted via EVs from infected cells, has opened a new window of opportunity to understand virus transmission and pathogenicity. Here, we used BTV, a complex multilayered virus, as a model to investigate the mechanism of virus-loaded EV formation and its effects on mammalian versus vector cells. Surprisingly, we discovered that in addition to large EVs (LEVs), BTV uses a small discrete size of EVs (SEVs) for efficient cell-to-cell transmission and that SEVs are enriched in the viral glycosylated NS3 protein when compared to particles associated with LEVs. Likewise, SEVs were also relatively more infectious, pointing to a role in transmission and pathogenicity. Our data identify two unique types of EVs associated with non-lytic release and the role of a non-structural viral glycoprotein in their formation.