Postmortem examination of neuropathogenesis in co-infected mice shows signs of brain herniation.

<p>(A) Representative mice infected with LCMV IC (left panel), MV IC (in NSE-CD46⁺ RAG-2 KO mice) (center panel), and both LCMV and MV (right panel) are shown exhibiting characteristic, post-mortem postures. (B) The scalp was removed from moribund co-infected mice or PBS treated controls to expose the skull; photographs were taken of the rear of the brain and cervical spinal cord. Note compression of white matter (cerebellum) against the posterior skull in co-infected animals. Mice were also examined for unilateral pupillary dilation (mydriasis) (seen here in mouse's left eye; white arrow). Photographs are representative of 5 to 10 mice per group.</p

LCMV-specific CD8⁺ T cells are found within the brains of co-infected mice.

<p>(A) T cells were isolated from brains of mice that were either uninfected (left panels), infected with MV IC alone (center panels), or infected with both LCMV IP and MV IC (right panels), as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002462#ppat-1002462-g003" target="_blank">Figure 3</a>. Cells were dual stained for CD8 (x-axis) and for one of two immunodominant LCMV epitopes (y-axis), using specific tetramers (GP33: top panels; NP396: bottom panels). (B) A standard ⁵¹Cr release assay was performed by incubating effector splenocytes from LCMV-infected mice (6 dpi) with uninfected (diamonds), MV-infected (squares), or LCMV-infected (triangles) target cells at the indicated ratios. Released ⁵¹Cr was measured in a gamma counter and specific release was calculated.</p

Tissue distribution of LCMV and MV in co-infected mice.

<p>(A) RNA was purified from the indicated tissues of co-infected mice at 4 dpi and amplified by quantitative RT-PCR for MV (black bars, scale on left) or LCMV (gray bars, scale on right). BD  =  below detection. (B) The extent of MV load was assessed in brains of mice infected with MV alone or with both MV and LCMV at 7 dpi. Data points represent individual mice, with averages shown as horizontal lines.</p

Edema, but not blood brain barrier, changes correlate with pathogenesis in co-infected mice and is dependent on CD8⁺ T cells.

<p>(A) Brains were removed 7 dpi from mice infected as indicated and weighed before and after dehydration in a vacuum oven. Total water weight was determined for each mouse and normalized to values from PBS IC mice; averages are shown as grey bars, with standard deviation indicated. Statistics were performed using the Wilcoxon Signed Rank test. #: p = 0.03; NS: not significant. (B) Mice, infected and treated as indicated, were anesthetized at 7 dpi and injected with Evan's Blue, followed by saline perfusion. Total Evan's Blue that diffused into the brain was determined by spectrophotometry following brain homogenization and clarification. Data were normalized to values from mice inoculated with PBS IC, and averages are shown as gray bars, with standard deviation indicated. Statistics were performed using the Wilcoxon Signed Rank test. NS: not significant. (C) Representative photomicrographs were taken from brain sections of mice with severe disease following co-infection with LCMV IP and MV IC. Evidence of choroid plexus inflammation (i), meningitis (ii), and hemosiderin deposition, indicative of capillary bleeding (iii), are shown. Original magnifications: A, B  =  640X; C  =  800X.</p

Morbidity following viral challenge.

<p>*Mice were inoculated with the specified dose via intracerebral inoculation in a volume of 30 ul. For group 5, virus was inactivated by exposure for 15 min to a UV light source and absence of infectious virus verified by plaque assay.</p><p>**Mice were inoculated with the specified dose via intraperitoneal inoculation in a volume of 200 ul.</p><p>***For interval studies, mice were infected with MV, rested for the indicated number of days, and then infected with LCMV.</p><p>****Morbidity was determined by either weight loss greater than 20% and/or seizures, ataxia or persistent tremors.</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

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

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