# Virion-Independent Extracellular Vesicle (EV)-Dependent Transmission of SARS-CoV-2 as a Potential New Mechanism of Viral RNA Spread in Human Cells

## TL;DR

This study explores how SARS-CoV-2 replicon RNA can be transferred between human cells via extracellular vesicles, suggesting a new mechanism for viral RNA spread.

## Contribution

The study demonstrates that extracellular vesicles can mediate SARS-CoV-2 replicon RNA transfer and replication in human cells.

## Key findings

- EVs from SARS-CoV-2 replicon cells transferred RNA to A549 cells, leading to replication and G-418 resistance.
- Paxlovid inhibited viral replication in a dose-dependent manner.
- Abnormal cellular changes persisted even after viral elimination.

## Abstract

The concentration of extracellular vesicles (EVs) in the peripheral blood of COVID-19 patients is increased. Nevertheless, their potential role in the transmission of infection remains unclear. This study was performed to determine whether EVs produced by the sub-genomic replicon system developed in Baby Hamster Kidney (BHK-21) cells could transfer SARS-CoV-2 replicon RNA, leading to the establishment of a viral replication system in human cells. Purified EVs from the SARS-CoV-2 sub-genomic replicon cell line BHK-21 were cultured with a naive human cell line. The success of EV-mediated transfer of SARS-CoV-2 replicon RNA and its productive replication was assessed using G-418 selection, a luciferase assay, immunostaining, and Western blot. We found that the A549 cell line cultured with EVs isolated from SARS-CoV-2 BHK-21 replicon cells developed G-418-resistant cell colonies. SARS-COV-2 RNA replication in A549 cells was confirmed by nano luciferase, Nsp1 protein. SARS-CoV-2 RNA replication causes massive morphological changes. Treatment of cells with the FDA-approved Paxlovid demonstrated a dose-dependent inhibition of viral replication. We isolated two human epithelial cell lines (gastrointestinal and neuroblastoma) and one vascular endothelial cell line that stably support high-level replication of SARS-CoV-2 sub-genomic RNA. Viral elimination did not revert the abnormal cellular shape, vesicle accumulation, syncytia formation, or EV release. Our study’s findings highlight the potential implications of EV-mediated transfer of replicon RNA to permissive cells. The replicon model is a valuable tool for studying virus-induced reversible and irreversible cellular reprogramming, as well as for testing novel therapeutic strategies for SARS-CoV-2.