Generating a Recombinant Virus with Heterologous Glycoproteins

Begin with a microplate containing host cells susceptible to vesicular stomatitis virus (VSV) infection.

Introduce a transfection mixture containing the genome plasmid, helper plasmids, and a transfection reagent that forms complexes with the plasmids. Incubate the cells to facilitate plasmid uptake.

The genome plasmid encodes a modified VSV genome in which the VSV glycoprotein (VSV-G) gene is replaced with one encoding an envelope glycoprotein (Env) from another virus.

The helper plasmids provide replication proteins, an RNA polymerase, and transient VSV-G for initial viral budding.

The polymerase transcribes the genome plasmid into viral RNA.

Then, the replication proteins generate subgenomic mRNAs to synthesize structural proteins, including the Env.

In the cytoplasm, recombinant VSV particles assemble and bud from the cell, incorporating the glycoproteins.

Collect the VSV-rich culture supernatant.

Apply cycles of freezing and thawing to lyse the cells and release trapped VSVs.

These recombinant VSVs encode essential replication genes along with heterologous envelope glycoproteins.