<i>In vivo</i> analysis of RABV-infected cells using Cre reporter mouse model.

<p>A) Timeline of mouse experiment. Cre reporter mice were infected intranasally (IN) with 10⁵ ffu RABV-Cre and sacrificed at the specified times. B) Weights of infected mice were monitored as a measure of disease throughout the experiment and demonstrate productive infection in all mice within this experiment. C) Brains collected at different time points post-infection were analyzed for the presence of EGFP-expressing cells in the following anatomical regions: olfactory bulb (OB), cerebral cortex (CC), cerebrum (CR), hippocampus (HIP), cerebellum (CB) and midbrain/hindbrain (MB-HB).</p

Microarray analysis of RABV-infected neurons isolated by FACS 3 months after infection indicates dysregulation of genes involved in nervous system function and cellular assembly.

<p>Cell suspensions prepared from whole mouse brains 3 months post-infection were sorted on a MoFlo cell sorter for EGFP+ (previously infected) and EGFP- (uninfected) cell populations. The 1248 transcripts differentially expressed between infected and uninfected cells (≥1.5 fold change, p<0.05) were analyzed by Ingenuity Pathway Analysis (IPA) to identify biological functions most significantly affected by the infection (significance predicted by p-value). Shown are the top ten most significant biological systems affected by the gene dysregulation, with the horizontal bars representing the negative log of their p-value (greatest significance at the top). Below each bar is the top three sub-categories affected by gene dysregulation in the respective categories. Each category/sub-category has the number of genes involved (up or down-regulated).</p

Neurons persist throughout the infected mouse brain long-after acute viral infection.

<p>The spread of the RABV infection was detected by identifying regions of EGFP fluorescence in different neuroanatomical regions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002971#ppat-1002971-g002" target="_blank">Figure 2C</a>) over a 6 month time course. Images are representative of 3–4 mice analyzed at each time point.</p

Characterization of Cre-expressing RABV.

<p>A) Genome of rabies virus (RABV) and the recombinant RABV expressing Enterobacteria phage P1 Cre recombinase (RABV-Cre) with a 5′ nuclear localization signal (shown in orange). B) Cre expression was confirmed by western blot analysis. Neuroblastoma cells were infected with either RABV-Cre, RABV expressing HIV-1 Gag (RABV-Gag), or mock infected (uninfected). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blotting with antibodies specific for Cre, RABV P or N, and actin. C) Viral growth kinetics were evaluated by multi-step growth curve assay in which BSR cells were infected at MOI 0.01 with either RABV or RABV-Cre, and viral titers determined from samples taken at the indicated time points post-infection. D) A schematic depicting the Cre-specific expression cassette in the Cre reporter mouse. In the absence of Cre, the chicken β-actin core promoter with a CMV enhancer (CAG) drives constitutive expression of membrane-targeted tandem dimer tomato (tdTomato) expression; EGFP is not expressed. After Cre-mediated excision of the tdTomato gene at the loxP sites, membrane-targeted enhanced green fluorescent protein (EGFP) is expressed. pA denotes polyadenylation sites. E) Cre functionality was evaluated <i>in vitro</i> by infecting primary fibroblasts isolated and cultured from Cre reporter mice for 96 hours with either RABV-Cre or RABV at MOI 20. EGFP and tdTomato labeling were detected by fluorescence microscopy (top) and flow cytometry (bottom) (using FITC and PE channels, respectively).</p

Neurons survive RABV infection and viral clearance.

<p>RNA was isolated from brains of mice at the indicated time points post-infection and assayed by real-time quantitative PCR for RABV genomic RNA (blue circles and line; left y-axis), RABV messenger RNA (black squares and line; left y-axis), and relative EGFP expression (green bars; right y-axis). Gene expression of all targets was normalized to the RPL13A (L13A) housekeeping gene. Data displayed was collected from 3 to 4 mice at each time point.</p

EGFP+ neurons are positive for RABV antigen.

<p>Brains were collected from Cre reporter mice fifteen days post-infection, cryosectioned, and EGFP+ regions compared to cell-specific labeling, A) NeuN (blue, neuronal nuclei antibody, 20× fluorescence imaging), B) GFAP (blue, astrocyte antibody, 40× confocal imaging), or C) RABV P antigen (purple) and DAPI nuclear stain (blue, 63× confocal imaging). White arrows in (C) indicate regions positive for RABV P.</p

Dysregulated genes and predicted effect on cell functions using Ingenuity Pathway Analysis.

*<p>Neur: growth of neurites; Cskel: organization of cytoskeleton; Cplsm: organization of cytoplasm; Micr: microtubule dynamics.</p>**<p>Increased: gene expression pattern predicts an increase in the specific function; Decreased: gene expression pattern predicts a decrease in the specific function; Affected: gene is involved in specific function, but unclear how it would influence it.</p

Recall response in mice after RABV-Gag prime and heterologous boost.

<p>(A) Schedule of prime-boost-challenge vaccinations. Mice were primed intramuscularly. 35 days later the mice were boosted. Mice were challenged intraperitoneally 33 days post prime. PBS mice were included as a negative control. 5 days after challenge, mice were euthanized and spleens harvested for analysis of the cellular response. (B–D) Each point is representative of splenocytes from one mouse (n = 5 per group). (B) The quantity of activated HIV-1 Gag-specific CD8⁺ T cells in the spleen was analyzed by flow cytometry and the percentage of cells are shown. Activated cells were determined by gating on CD62L^lo cells and HIV-1 Gag-specific cells were determined by tetramer staining against the H2^d restricted AMQMKLETI epitope. (C–E) The functionality of the CD8⁺ T cells was measured by intracellular cytokine staining for (C) IFNγ⁺, (D) TNFα⁺, and (E) IFNγ⁺TNFα⁺ cells after stimulation of the cells with AMQMKLETI peptide. Statistical analysis was performed using One-Way ANOVA. All comparisons to PBS are statistically significant. Results shown are presented as the mean. **p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Analysis of the Gag-specific CD8⁺ T Cells in the spleen after VSV-Gag prime and heterologous boost.

<p>(A) Schedule of prime-boost vaccinations. Mice were primed intramuscularly. 35 days later the mice were boosted. Mice mock-immunized with PBS were included as a negative control. 5 days after boost, mice were euthanized and spleens harvested for analysis of the cellular response. (B–D) Each point is representative of splenocytes from one mouse (n = 5 per group). (B) The quantity of activated HIV-1 Gag-specific CD8⁺ T cells in the spleen was analyzed by flow cytometry and the percentage of cells are shown. Activated cells were determined by gating on CD62L^lo cells and HIV-1 Gag-specific cells were determined by tetramer staining against the H2^d restricted AMQMKLETI epitope. (C–E) The functionality of the CD8⁺ T cells was measured by intracellular cytokine staining for (C) IFNγ⁺, (D) TNFα⁺, and (E) IFNγ⁺TNFα⁺ cells after stimulation of the cells with AMQMKLETI peptide. Statistical analysis was performed using One-WayANOVA. Results shown are presented as the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Recall response in mice after NDV-Gag prime and heterologous boost.

<p>(A) Schedule of prime-boost-challenge vaccinations. Mice were primed intramuscularly on Day 0 and 6. 35 days later the mice were boosted. Mice were challenged intraperitoneally 33 days post prime. PBS mice were included as a negative control. 5 days after challenge, mice were euthanized and spleens harvested for analysis of the cellular response. (B–D) Each point is representative of splenocytes from one mouse (n = 5 per group). (B) The quantity of activated HIV-1 Gag-specific CD8⁺ T cells in the spleen was analyzed by flow cytometry and the percentage of cells are shown. Activated cells were determined by gating on CD62L^lo cells and HIV-1 Gag-specific cells were determined by tetramer staining against the H2^d restricted AMQMKLETI epitope. (C–E) The functionality of the CD8⁺ T cells was measured by intracellular cytokine staining for (C) IFNγ⁺, (D) TNFα⁺, and (E) IFNγ⁺TNFα⁺ cells after stimulation of the cells with AMQMKLETI peptide. Statistical analysis was performed using one-WAY ANOVA. All comparisons to PBS are statistically significant. Results shown are presented as the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p