Green fluorescent protein expressed by recombinant pseudorabies virus as an in vivo marker for viral replication.

We isolated and characterized a pseudorabies virus (PrV) mutant expressing an engineered green fluorescent protein (GFP) optimized for expression in human cells. The GFP DNA was inserted in the non-essential glycoprotein 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 FITC fluorescence. In fixed samples detection sensitivity was increased by immunofluorescence using an anti-GFP antibody. Specifically labelled PrV mutants have been used successfully as transsynaptic circuit tracers 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). Availability of this recombinant allows the study of even more complex interactions using differentially labelled PrV mutants, and provides a means to monitor viral replication and spread without destruction of the cell.