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

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

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

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

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