# Extracellular Vesicles Released by Bovine Alphaherpesvirus 1-Infected A549 Cells May Limit Subsequent Infections of the Progeny Virus

## TL;DR

Bovine alphaherpesvirus 1-infected cells release extracellular vesicles that block virus spread and boost immune responses, potentially offering a new antiviral strategy.

## Contribution

Discovery that extracellular vesicles from infected cells inhibit viral replication and enhance antiviral immunity through novel mechanisms.

## Key findings

- Extracellular vesicles from BoAHV-1-infected cells contain 59 viral proteins and inhibit viral binding to cells.
- EDVs stimulate IFN-α and IFN-β production in A549 cells and IFN-γ in rabbit trigeminal ganglia neurons.
- Combining EDVs with Acyclovir enhances antiviral efficacy and may serve as a drug delivery vector for TG tissues.

## Abstract

Bovine alphaherpesvirus 1 (BoAHV-1) is a promising oncolytic virus that can infect the human lung carcinoma cell line A549. In an effort to adapt the virus to grow more rapidly in these cells through the serial passaging of viral progeny, we were unsuccessful. Here, we found that extracellular vesicles (EVs) secreted by BoAHV-1-infected A549 cells (referred to as EDVs) contain 59 viral proteins, including both viral structure proteins (such as gC and gD) and viral regulatory proteins (such as bICP4 and bICP22), as identified via a proteomic analysis. These EDVs can bind to and enter target cells, inhibit viral particles binding to cells, and stimulate the production of IFN-α and IFN-β in A549 cells. When EDVs are inoculated into rabbits via either the conjunctival sacs or intravenously, they can be readily detected in neurons within the trigeminal ganglia (TG), where they reduce viral replication and promote the transcription of IFN-γ. Furthermore, incorporation of the known anti-herpesvirus drug Acyclovir (ACY) into the EDVs leads to synergistically enhanced antiviral efficacy. Collectively, the EDVs exhibit antiviral effects by blocking viral binding to target cells and stimulating the innate immune response, thereby leading to the failure of the serial passaging of viral progeny in these cells, and these EDVs may serve as a promising vector for delivering drugs targeting TG tissues for antiviral purposes.