Generating Infectious Virus Particles Containing a BACmid-Derived Viral Genome

Begin with a BACmid, a bacterial artificial chromosome that can replicate like a plasmid inside bacteria.

The BACmid contains the entire genome of the vaccinia virus with targeted modifications, but it is not infectious as it requires viral proteins to function.

Take a helper virus, which is an attenuated vaccinia virus encoding the gene for EGFP.

Infect epithelial cells with the helper virus and simultaneously transfect with the BACmid.

The virus expresses EGFP and provides the viral proteins, facilitating transcription, replication, and packaging of the BACmid-derived genome into virions, which infect nearby host cells.

Under a fluorescence microscope, select an EGFP-negative plaque to ensure it contains cells infected only with BACmid-derived viruses.

Collect the cells from the selected plaque and subject them to a freeze-thaw cycle, followed by sonication, to release intracellular virus particles.

Centrifuge and collect the supernatant containing infectious BACmid-derived virus particles for further use.