FIG. 4.

Viral genetic requirements of in vitro transneuronal spread. Explants were infected with PRV Becker, PRV Bartha, GS442 (a complemented gD null virus that expresses GFP), or HF22A (a complemented gB null virus). (A) Wide-field images of neurons that are fixed and stained with anti-VP5 antibody and Hoechst 33342 at 24 h postinfection. Only dissociated cells inside the chamber are shown. The same field of neurons is shown in the anti-VP5 panel as in the Hoechst panel. Three chambers were used for each PRV strain. (B) Quantitation of neurons infected inside chambers. Neurons positive for anti-VP5 (α-VP5) staining were scored as infected. (C) Quantitation of spread via titer determination. The medium in the chamber was harvested at 24 h postinfection, and the titer was determined for PRV plaques on PK15 cells. Three chambers were used for each PRV strain. Scale bar, 20 μm.