Conditional Transneuronal Tracing with PRV Figure 1. PRV Genome. The genomic organization of PRV-Becker, PRV-Bartha and the recombinants prepared for this study are illustrated. The basic organization of the PRV genome is illustrated at the top of the figure (A). Viral DNA contains unique long (UL) and unique short (US) segments flanked by internal and terminal repeat sequences. The BamH1 restriction map of PRV (B) illustrates the location of the portions of the viral genome engineered to express transgenes in PRV-263 and PRV-267. These regions of the restriction map are expanded in C to illustrate the location of the genes in segments 1 and 7 (C). Boxes represent individual genes, with the formal name indicated above the box and the common name, where appropriate, indicated within each box. The recombinants prepared for this analysis are derived from the PRV-Bartha genome, which contains a large deletion in the US segment. The genes eliminated by this deletion reduce virulence and restrict viral transport through circuits to the retrograde direction. The Brainbow cassette and Cre were inserted into the gG (US4) locus to create PRV-263 and PRV-267, respectively. The organization of the Brainbow 1.0L cassette is illustrated in section D of the figure. Paired loxP and lox2272 sites are positioned within the cassette such that recombination at loxP sites eliminates the dTomato and mCerulean genes to liberate expression of EYFP and recombination at lox2272 sites eliminates the red reporter gene to liberate expression of mCerulean. It is important to note that Cre only cuts at like pairs (e.g., loxP:loxP or lox2272:lox2272) and that the cassette (intact or recombined) will only express one reporter. PRV-267 also carries mRFP as part of a fusion gene at the VP26 (UL35) locus to produce a unique marker of the surface capsid protein VP26. Construction of PRV-267 is described in the Materials and Methods. Construction of PRV-263 has been reported [17]. doi:10.1371/journal.pone.0021141.g001 Immunoperoxidase localizations The invasive profiles of the recombinants were first determined by immunoperoxidase localization of infected neurons. Coronal sections at a frequency of 210 mm through the brain and horizontal sections at a frequency of 16 mm through the spinal cord were processed from each case. Viral immunoreactivity was detected with a rabbit polyclonal antiserum (Rb133) generated against acetone-inactivated virus [27]. This antiserum recognizes epitopes on all virally encoded proteins and was used at a 1:10,000 dilution in conjunction with affinity purified, biotinylated donkey anti-rabbit secondary antibody (1:200; Jackson ImmunoReseach Laboratories, Inc.; West Grove, PA) and Vectastain Elite avidin- biotin reagents (9 ml of each reagent combined 90 minutes before tissue incubation; Vector Laboratories; Burlingame, CA). Diami- nobenzidine (DAB) was used as a substrate for the immunoper- oxidase reaction; tissue was incubated in the DAB solution for 10 minutes prior to addition of 35 ml of H2O2/100 ml DAB solution to catalyze the reaction, and the reaction was terminated 3 minutes after H2O2 addition by repeated rinses in sodium phosphate buffer. Processed sections were mounted on Superfrost PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21141