Efficacy of virus inoculation and reactivation in mice.

(A-C) Efficacy of virus inoculation. (A) Western blot showing the presence of ICP4 in trigeminal ganglia (TGs) from HSV1-M (n = 6) and CTRL-M (n = 2) sacrificed 4 days p.i.; tubulin expression level was used as sample loading control. (B) Lip, TG, hippocampus (HP), cortex (CTX) and cerebellum (CB) from HSV1-M were homogenized as described in Methods, and incubated on VERO cells for 3 h, then replaced with complete RPMI. Four days p.i., cell supernatants were harvested and then assayed on VERO cells by standard plaque assay (SPA, n = 2 for each tissue) and Reed and Muench method (TCDI50, n = 2 for each tissue) to evaluate virus titer. Scatter dot plot shows the individual and mean values of virus titer (log10 PFU/ml) assayed by SPA (blue dots) and TCID50 (purple dots). (C) In Cell Western (ICW) assay, upper image: after 3h of incubation with the indicated serial dilutions of TG-derived supernatants (TG-derived supernatants, n = 2, obtained as described in B), VERO cells were left for 72 h with RPMI 2% FBS and then fixed and stained with anti-gB antibody (green, upper wells); lower image: the same staining was performed on VERO cells infected for 24 h with stock HSV-1 or mock solution at the indicated dilutions (Infected VERO cells). Arrowheads indicate representative gB+ foci of infection. (D) TG and the indicated brain tissues were harvested from 4 HSV1-M sacrificed 24h after the 3rd TS, homogenized as described in Methods, incubated on VERO cells for 3 h, then replaced with complete RPMI. Four days p.i., cell supernatants were harvested and then assayed on VERO cells by SPA. Scatter dot plot shows the individual and mean values of virus titer (log10 PFU/ml) assayed by SPA (blue dots).