Recovery of Zika Virus from In Vitro-Transcribed RNA

Begin with tubes containing a suspension of Vero cells.

Add water to the control sample, and introduce an in vitro transcribed (IVT) RNA into the experimental sample.

The RNA represents the full-length genome of the Zika virus.

Transfer each sample into electroporation cuvettes.

Apply electroporation.

A brief electrical pulse disrupts the host cell membrane and creates transient pores, allowing the RNA to enter the cells.

Add a medium to support cell recovery.

Transfer the cells into flasks containing a medium.

Incubate to allow the viral RNA to drive protein synthesis and genome replication, leading to the assembly of new virions.

The virions are released and spread to neighboring cells.

The increasing intracellular viral burden eventually causes host cell death.

Monitor the cultures under a microscope.

Increased cell death in the experimental sample compared with the control confirms the recovery of infectious virus from IVT RNA.

One or two days prior to electroporation, prepare one T182 flask of Vero cells per construct to be recovered.

Include a T182 flask to be used as a mock electroporation control. When cells are at 70 to 80% confluency, warm DMEM containing 15% FBS and 10 millimolar heap