Fig 5.

Impaired reovirus binding to sialic acid does not limit infection of sites in the brain. (A–D) Two-day-old WT mice were inoculated IC with phosphate-buffered saline (mock) or 1,000 PFU of reovirus strain T3SA+ or T3SA−. Mice were euthanized at 7 days post-inoculation, and brain tissue and whole blood were collected. (A) Titers of virus in homogenized left-brain hemispheres and blood were determined by plaque assay. Each symbol represents the viral titer from an individual animal. Brain, N = 29/29 (T3SA+/T3SA−); blood, N = 19/20 (T3SA+/T3SA−). Error bars indicate SEM. Values that differ significantly from T3SA+ by unpaired t test are indicated (****P < 0.0001). Dotted line indicates limit of detection. (B–D) Right-brain hemispheres (with contralateral hemisphere viral loads between 3.4e⁸ and 7.3e⁹) were fixed with formalin, embedded in paraffin, and sectioned sagittally. Tissue sections were probed for reovirus RNA by HCR, counterstained with Hoechst dye, and imaged using a Lionheart FX automated imager. (B) Representative images are shown for the indicated brain regions. Reovirus RNA is depicted in green; nuclei are depicted in blue. Scale bar, 1,000 µm. (C and D) Reovirus infection in established regions of interest. Infection foci (HCR-positive) from each region (C) or subregion (D) of mock-infected and reovirus-infected sections were enumerated using the Spot Detector tool within Icy software. Data are presented as the percentage of infected cells, wherein a reovirus-positive cell was determined by signal intensity greater than background defined using a mock-infected brain. N = 5/7 (T3SA+/T3SA−). Error bars indicate SEM. Values that differ significantly from T3SA+ by Sidak’s multiple comparisons test are indicated (*P < 0.05 and **P < 0.005). DPall, dorsal pallium; MTt, collicular midbrain tectum; MPall, medial pallium; PH, pontine hindbrain; PPH, prepontine hindbrain; Th, thalamus; Thy+PHy, hypothalamus; and SPall, subpallium.