Ddost overexpression strengthens antiviral immune responses of mice suffering from HSE.

(A, B) Stable mouse Neuro-2a (N2a) cells expressing Flag-Ddost and control N2a cells expressing empty vector (Vec) were prepared by lentiviral transduction and puromycin (1 μg/mL) selection for 10 days. HSV-1 (MOI = 4) was added to the cells for the indicated time, and cell lysates were collected for immunoblotting analysis (A) or qPCR (B). Data displayed are the mean ± SD (n = 3). **P P t test). (C) Stably expressing Flag-Ddost N2a cells and control cells (Vec) were infected with HSV-1 (MOI = 2) for 24 hours. HSV-1 replication was determined by plaque assays. Data displayed are the mean ± SD (n = 3). **P t test). (D) The expression of ectopic and endogenous Ddost in different brain regions 14 days after AAV-Ddost injection was determined by immunoblotting analysis. (E) Schematic diagram of AAV stereotactic injection and HSV-1 delivery into the mouse hippocampus region. (F) Ddost-overexpressing and control mice (n = 8 for each group) were intracranially injected with GFP-HSV-1 (2×105 PFU per mouse). Mouse survival was monitored, and the survival rates were calculated and presented as Kaplan–Meier curves. (G-J) Ddost-overexpressing mice were injected with HSV-1 (3×104 PFU per mouse) for 24 hours. The hippocampus was separated and analyzed by qPCR for the indicated genes. The results were normalized to Gapdh. Data displayed are the mean ± SD (each dot represents the result from one mouse). *P P P 4 PFU per mouse) for 4 days (n = 5 for each group). Frozen sections (10 μm) of the hippocampus were stained with anti-Iba-1 antibody and Cy3-labeled secondary antibody. Representative confocal images are shown. Scale bar, 200 μm.