GREEN FLUORESCENT PROTEIN EXPRESSED BY RECOMBINANT PSEUDORABIES VIRUSAS AN IN-VIVO MARKER FOR VIRAL REPLICATION

We isolated and characterized a pseudorabies virus (PrV) mutant expres sing an engineered green fluorescent protein (GFP) optimized for expre ssion in human cells. The GFP DNA was inserted in the non-essential gl ycoprotein G (gG) gene of the attenuated PrV strain Bartha. The coding sequence was cloned in frame behind the first seven codons of the gG gene under control of the strong gG promotor. On excitation with blue light, live cells infected with the recombinant PrV B80eGFP exhibited bright fluorescence when examined microscopically using filters for FI TC fluorescence. In fixed samples detection sensitivity was increased by immunofluorescence using an anti-GFP antibody. Specifically labelle d PrV mutants have been used successfully as transsynaptic circuit tra cers for definition of central command neurons in the brain (Jansen et al., 1995. Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response. Science 270, 644-646). Availab ility of this recombinant allows the study of even more complex intera ctions using differentially labelled PrV mutants, and provides a means to monitor viral replication and spread without destruction of the ce ll. (C) 1997 Elsevier Science B.V.