A method of alphavirus replicon particle titration based on expression of functional replicase/transcriptase.

Alphavirus replicon particles are being exploited for a variety of purposes both in vitro as gene expression vectors, and in vivo as vaccines or gene therapy vectors. There is a need for a simple and universal method of titration of replicon particles that is independent of expression of the foreign protein. We devised a method that uses modified vaccinia virus Ankara (MVA) as an indicator virus, to deliver a Venezuelan equine encephalitis virus (VEE) defective helper RNA encoding green fluorescent protein (GFP). Co-infection of cells with the MVA-based indicator and Venezuelan equine encephalitis virus replicon particles (VRP) results in expression of the GFP gene. VRP titer is readily determined by counting fluorescent cells.