Rescue of Infectious Virions from Cells Transfected with Bacterial Artificial Chromosome-Derived Synthetic RNA

Begin with an adherent, virus-permissive cell monolayer.

Wash the cells, then add a dissociating agent to detach them.

Centrifuge and discard the supernatant. Wash the cells to remove the residual medium.

Resuspend the cells in buffered saline, then transfer the cells to a cuvette.

Introduce RNA transcribed from a full-length Japanese encephalitis virus (JEV) cDNA clone carried in a bacterial artificial chromosome.

Apply electric pulses to transiently permeabilize the cell membrane, allowing RNA entry.

Let the cells recover, then transfer them to a culture medium.

The introduced RNA mimics native viral RNA to produce new virions.

Serially dilute the cells and plate them onto fresh cell monolayers.

Incubate to allow the virions to infect the neighboring cells.

Overlay with agarose media and incubate. The medium forms a semi-solid layer, restricting viral spread to nearby cells.

The infected cells lyse and form discrete plaques, confirming successful rescue of infectious JEV.

Start with BHK-21 cells grown for 24 hours in 150 millimeter dishes plated at 3 million cells per dish. Rinse the cell monolayer with cold solution A and then detach them by trypsinization. Collect the cells by centrifugati