Background: Getah virus (GETV) is a mosquito-borne alphavirus of veterinary importance with potential zoonotic relevance. Extracellular vesicles (EVs) can facilitate intercellular transfer of viral components, but their involvement in GETV infection remains unclear.

Methods: Exosome-enriched EV fractions were isolated from supernatants of GETV-infected HeLa, BHK21, and PK15 cells by ultracentrifugation followed by CD9 immunoaffinity enrichment (HeLa) or iodixanol density gradient fractionation (BHK21/PK15). EV-associated viral RNA and infection-related readouts were analyzed using RT-PCR, RT-qPCR, immunoblotting, immunofluorescence, transmission electron microscopy, and uptake assays.

Results: Exosome-enriched EV fractions were enriched for canonical EV markers and were associated with genome-spanning GETV RNA. Exposure of recipient cells to these fractions resulted in EV uptake, increased intracellular GETV RNA levels, and viral protein expression. RNase protection assays showed that the RNA signal was resistant to RNase treatment unless membranes were disrupted, consistent with membrane-protected RNA. In donor HeLa cells, GW4869 treatment reduced EV recovery and EV-associated GETV RNA levels and decreased transmission-related readouts in recipient cells, while having limited effects on cell-free virus replication in donor cells.

Discussion: Together, these findings support the existence of an EV-associated route that may contribute to GETV transmission-related processes in vitro. However, complete physical separation of EVs from virions and the mechanism of genome delivery require further validation.

Keywords: Getah virus; alphavirus; exosome-enriched EVs; extracellular vesicles; intercellular spread.