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.
Gangolli
Seema S
SS
Wyeth Vaccines, Viral Vaccine Discovery Research, Pearl River, NY 10965, USA.
Vasilakis
Nikolaos
N
Kovacs
Gerald R
GR
Zamb
Timothy J
TJ
Kowalski
Jacek
J
D000818:Animals; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D003673:Defective Viruses / Q000235:genetics*; D004666:Encephalitis Virus, Venezuelan Equine / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D012093:Replicon / Q000235:genetics*; D014616:Vaccinia virus / Q000235:genetics; D014709:Vero Cells
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D012321:DNA-Directed RNA Polymerases