Replication and spread of virus in the brain after intranasal infection with NSV.

<p>Representative thalamus sections from the brains of at least 3 WTB6, GRKO and GKO mice 3, 5 and 7 days after intranasal infection with 10⁵ pfu NSV were stained with rabbit polyclonal antibody to NSV followed by biotinylated antibody to rabbit IgG, avidin-peroxidase and DAB. NSV (brown staining) was observed in neurons (arrows) at 3 (A, D, G) and 5 (B, E, H) days after infection in all strains of mice. At 7 days after infection NSV antigen was decreased in WTB6 (C), but not GRKO (F) or GKO (I) mice and neurons (circled) were degenerating. Insets show 2-fold magnified images of the NSV-positive neurons indicated by the arrows or circles. X300. Scale bar = 100um.</p

Mortality and virus replication after intranasal NSV infection.

<p>(A) 6-9 week-old WTB6 (<i>N</i> = 11), GKO (<i>N</i> = 24) and GRKO (<i>N</i> = 23) mice were infected intranasally with 10⁵ pfu NSV and followed daily for mortality which was similar. <i>P</i> = 0.47. At 3, 5, and 7 d after infection, brains (B), cervical (C) and lumbar (D) spinal cords from 3 WTB6, GKO or GRKO mice were assayed for infectious virus by plaque formation on BHK cells. LOD = limit of detection. * <i>P</i> < 0.02; t test.</p

Immunohistochemical staining for IFN-gamma R2 in brains of WTB6 and GRKO mice.

<p>The hippocampus (A,B) and basal forebrain (C,D) of NSV-infected WTB6 (A,C) and GRKO (B,D) mice were examined 7 days after infection for expression of IFN-gamma R2. Frozen sections of the brains from 2 animals/group were immunostained with hamster monoclonal anti-IFN-gamma R2. IFN-gamma R2 was intensely expressed by cells in the brains of WTB6 mice (arrows). In GRKO mice that have a disruption in the IFN-gamma R1 gene, expression was less (arrows), but the distribution was similar. X200 Scale bar = 100 um.</p

Immunohistochemical staining for MHC class I.

<p>Frozen sections from the thalamus of WTB6 (A), GRKO (B) and GKO (C) mice were stained with mouse monoclonal anti-H-2K^b/H-2D^d for identification of MHC class I antigen-positive cells 7 days after intranasal infection with 10⁵ pfu NSV. Representative sections are shown. X250. Scale bar = 100 um. (D) Quantification of MHC class I-positive cells/0.16 mm² (unit area) in thalamus tissue from 2 mice in each group. Photomicrographs of 5-7 areas were taken (250X magnification) and numbers of stained cells were counted. Each bar indicates the mean +/- SEM. *** P < 0.001; t test.</p

Cytokine and chemokine mRNA induction after treatment of WTB6 and GRKO MEFs with IFN-gamma.

<p>Total RNAs were collected from WTB6 and GRKO MEFs at various times after IFN-gamma treatment (100 U/mL) and assessed for levels of mRNA. (A) Changes in IL-6 and TNF-alpha mRNAs as determined by RT-PCR. Results representative of 3 experiments are shown. (B) Changes in levels of CXCL9 (diamonds) and CXCL10 (circles) mRNAs in WTB6 (solid lines) and GRKO (dashed lines) MEFs by qRT-PCR. (C) Changes in levels of CCL1 (squares), CCL2 (triangles) and CCL5 (inverted triangles) mRNAs in WTB6 (solid lines) and GRKO (dashed lines) MEFs by qRT-PCR. Data were normalized to GAPDH expression and are presented as mean +/- SEM of triplicate samples and are representative of three experiments. </p

Immunohistochemical staining for nonstructural protein 3 in the piriform cortex.

<p>Brains from WTB6 (A), GRKO (B), and GKO (C) mice were examined 7 days after intranasal infection with 10⁵ pfu NSV. Representative sections from at least 3 mice were stained with a rabbit polyclonal antibody to nsP3 followed by biotinylated antibody to rabbit IgG, avidin-peroxidase and DAB. nsP3-positive cells were more frequent in GRKO and GKO mice. Insets contain magnified versions of encircled cells. X400. Scale bar = 100 um.</p

Immunohistochemical staining and quantification of inflammatory cells in the thalamus.

<p>Brains from WTB6, GRKO and GKO mice 7 days after intranasal infection with 10⁵ pfu NSV were examined for CD3⁺, CD4⁺, perforin⁺, B220⁺ and Iba-1⁺ cells. Frozen sections of the thalamus from 2 animals per group were immunostained with rabbit polyclonal anti-CD3, rabbit polyclonal anti-CD4, rat monoclonal anti perforin, rat monoclonal anti-B220 and rabbit polyclonal anti Iba-1. (A) Immunohistochemical staining for inflammatory cells. Representative sections are shown. X200. Scale bar = 200 um. (B) Photographs of 13-16 unit areas (0.25 mm²)/group were taken (X200 magnification) and numbers of brown stained immune cells were counted. Each bar indicates the mean and SEM. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001; t test.</p

Immunohistochemical staining for MHC class II.

<p>Frozen sections from the thalamus (A-C, upper panels) and brainstem (D-F, lower panels) of WTB6 (A,D), GRKO (B,E) and GKO (C,F) mice were stained with mouse monoclonal anti-I-A/I-E for identification of MHC class II antigen-positive cells 7 days after intranasal infection with 10⁵ pfu NSV. X250. Scale bar = 100 um. (G) Quantification of MHC class II-positive cells/0.16 mm² tissue in thalamus and brainstem from 2 animals in each group. Photomicrographs of 5-6 areas of thalamus and 10 areas of brainstem were taken and stained cells counted. Each bar indicates the mean and SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001; t test.</p

Quantification of cytokine mRNA levels in the brain.

<p>Quantitative RT-PCR was used to measure the levels of IFN-gamma (A), IL-1alpha (B), TNF-alpha (C), IL-2 (D), IL-12alpha (E), IL-6 (F), IL-4 (G), IL-10 (H), IL-13 (I) and IL-17A (J) mRNAs in the brains of uninfected (mock) and NSV-infected WTB6, GKO and GRKO mice. Cytokine mRNAs were normalized to beta-actin mRNA. Data are from 3 animals from each group at each time point and 2-4 experimental replicates. Bars indicate the mean and SEM for each group. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, **** <i>P</i> < 0.0001; ANOVA.</p

Effect of IFN-gamma on NSV infection of mouse embryo fibroblasts from GRKO and WTB6 mice.

<p>The responses of MEFs from WTB6 and GRKO mice to treatment with 100 U recombinant rat IFN-gamma were assessed over 24-48h. (A) Immunoblot to detect phosphorylation of STAT1 tyrosine 701 and changes in STAT1 protein levels. A representative of 3 experiments is shown. (B) RT-PCR to detect changes in transcription of antiviral genes 2-5 oligoadenylate synthase (OAS) and zinc finger antiviral protein (ZAP). Data shown are representative of 3 experiments. (C) Cell viability as determined by trypan blue exclusion 24h after infection with NSV (MOI = 5) in the presence and absence of IFN-gamma. Data are averaged from 2 experiments and plotted as mean +/- SEM. **** P < 0.0001; ANOVA (D) Effect of IFN-gamma on production of infectious virus by WTB6 (squares) and GRKO (triangles) MEFs after NSV infection with (dashed line) and without (solid line) IFN-gamma pretreatment. Data are from three experiments and plotted as mean +/- SEM. *** P < 0.001; ANOVA. </p