Triple-label immunofluorescent staining for macrophage marker CD68 (green, Alexa 488), SIV envelope protein (red, Cy5), and astrocyte marker GFAP (blue, Alexa Fluor 555) shows infected macrophages in a microglial nodule from a CD8⁺ T-cell-depleted rhesus macaque with SIVE (macaque M139). Yellow indicates co-localization of CD68 and SIV. Aqua would show co-localization of SIV and GFAP, however no productively infected astrocytes were detected. Scale bar, 20 μm.

SIV RNA of eight CD8-depleted rhesus macaques infected with SIV/DeltaB670. Based on histological findings, macaques were retrospectively classified at necropsy for presence of SIV encephalitis. Plasma viral load (a) in macaques that developed encephalitis was significantly higher at 1 and 3 weeks after infection compared to macaques that did not develop encephalitis. Between 6 and 8 weeks after infection, higher CSF viral load (b) distinguishes macaques that developed encephalitis from macaques that did not develop encephalitis. a: Median longitudinal plasma SIV RNA for the three macaques with SIVE are shown in black and the five macaques without encephalitis are shown in gray. Plasma SIV RNA of macaques that developed encephalitis was significantly higher than macaques without encephalitis at 1 and 3 weeks after infection. *P < 0.05. b: Median longitudinal CSF SIV RNA for the three macaques with SIVE are shown in black and the five macaques without encephalitis are shown in gray. CSF SIV RNA of macaques that developed encephalitis was higher than macaques without encephalitis throughout the length of infection, especially during the end stages of infection. Because of unavailable CSF samples, statistical analyses could not be completed for all time points. c: Median CSF SIV RNA for macaques with SIVE (black) and macaques without encephalitis (gray) in weeks before death. Macaques that develop SIVE have more virus in CSF than macaques that do not develop encephalitis in the weeks leading up to death.

Double-label immunofluorescent staining for macrophage marker CD68 or T-cell marker CD3 and SIV envelope protein shows macaques with SIVE have more infected macrophages in the spinal cord but less infected macrophages in the thymus and lung than macaques without encephalitis. Images such as these were used to count the number of infected macrophages and T cells for tabulation in Figure 6. A–D, F, and G: Small images on the left illustrate individual channels for CD68 (red, Cy5) and SIVgp110 (green, Alexa 488). The large image on the right shows a merged image of the red and green channel with yellow indicating co-localization of CD68 and SIVgp110. E and H: Small images on the left illustrate individual channels for CD3 (red, Cy5) and SIVgp110 (green, Alexa 488). The large image on the right shows a merged image of the red and green channel with yellow indicating co-localization of CD3 and SIVgp110. A: A histological section of spinal cord from a macaque with SIVE (M139). Macrophages are the predominant SIV-infected cell with rare noninfected T cells. B: A histological section of spinal cord from a macaque without encephalitis (M158). Macrophages are less abundant and not infected with SIV. C: A histological section of thymus from a macaque with SIVE (M144). There are few SIV-infected cells and less macrophages than in macaques without encephalitis. D and E: Histological sections of thymus from a macaque without encephalitis (M135). Some of the infected cells in the thymus of macaques without encephalitis were macrophages, fewer were T cells, but the majority of SIV-infected cells did not co-label with either CD68 or CD3. F–H: Histological sections of lung from a macaque without encephalitis (M135) (G and H) and a macaque with SIVE (M139) (F). Some of the infected cells in the lung of macaques without encephalitis were macrophages, but the majority of cells did not co-label with either CD68 or CD3. Macaques with SIVE had few SIV-infected cells in their lungs. Scale bars, 20 μm.

Analysis of post- and presynaptic proteins and macrophages in the brains of eight CD8-depleted rhesus macaques infected with SIV/DeltaB670 at necropsy. Based on histological findings, macaques were retrospectively classified postmortem as having SIV encephalitis (enc), without encephalitis (no enc), or noninfected controls (con). Each indicated brain region was immunostained for MAP-2 (a), synaptophysin (b), and CD68 (c) and visualized by immunofluorescent confocal microscopy. Ten microscopic fields in each brain region were quantified for each animal and marker. MFI multiplied by the number of pixels (area) covered by fluorescence was quantified in each brain region using immunofluorescent confocal laser microscopy as described in Materials and Methods. For the basal ganglia region, average MFI and pixels for MAP-2 and synaptophysin were compared to macaques without encephalitis and reported as average MFI* area because of limited availability of basal ganglia tissue from noninfected macaques. The black bars represent the median number of infected cells enumerated for each group, whereas each dot represents the enumeration from an individual field. Macaques with SIVE showed less postsynaptic protein (MAP-2 staining) but not presynaptic protein (synaptophysin staining) in cortical regions than macaques without encephalitis. Macaques with SIVE showed more CD68 staining in cortical regions than macaques without encephalitis. a: MAP-2. MAP-2, postsynaptic protein, staining is decreased in the brains of macaques with SIVE. b: Synaptophysin, presynaptic protein, staining is similar in the brains of macaques with and without encephalitis. c: CD68. CD68, a macrophage/microglia-associated protein, staining is significantly increased in the brain regions of macaques with SIVE. * P < 0.05, **P < 0.01, ***P < 0.001.

Longitudinal peripheral blood counts for CD4⁺ lymphocytes (a), CD8⁺ lymphocytes (b), and monocytes (c) of eight CD8-depleted rhesus macaques infected with SIV/DeltaB670. Based on histological findings, macaques were retrospectively classified at postmortem for presence of SIV encephalitis. CD4⁺ and CD8⁺ lymphocyte profiles of animals that did or did not develop encephalitis were similar; however, peripheral blood CD4⁺CD29⁺ T-helper lymphocytes (d) decreased at 2 weeks after infection in macaques that developed encephalitis. Macaques that developed encephalitis had lower average white blood cell counts before infection or CD8 depletion. a: Peripheral blood absolute CD4⁺ lymphocyte counts decreased throughout the course of infection. Median peripheral blood absolute CD4⁺ lymphocyte counts for the three macaques with SIVE are shown in black and the five macaques without encephalitis are shown in gray. b: Peripheral blood CD8⁺ lymphocytes were suppressed for 2 to 4 weeks in macaques administered cM-T807 during primary infection. Median peripheral blood absolute CD8⁺ lymphocyte counts for the three macaques with SIVE are shown in black and the five macaques without encephalitis are shown in gray. The asterisk at day 14 after infection indicates a statistically significant difference in the peripheral blood absolute CD8⁺ lymphocyte count between macaques with and without encephalitis. *P < 0.05. All other time points are not statistically different. c: Peripheral blood absolute monocyte counts were not statistically different between macaques with and without encephalitis. Median peripheral blood absolute monocyte counts for the three macaques with SIVE are shown in black and the five macaques without encephalitis are shown in gray. d: Peripheral blood CD4⁺CD29⁺ T-helper lymphocytes decreased earlier in macaques that developed SIVE. Median percentage of peripheral blood T-helper lymphocytes (% CD4⁺CD29⁺/total % CD4⁺ cells) for the three macaques with SIVE are shown in black and the five macaques without encephalitis are shown in gray.

Analysis of blood monocyte SIV DNA and SIV p27 production in MDMs and nonadherent PBMCs from CD8⁺ T-cell-depleted rhesus macaques during SIV/DeltaB670 infection. Based on histological findings, macaques were retrospectively classified at necropsy for presence of SIV encephalitis. a: Peak number of SIV DNA copies/ml blood in CD14⁺ blood monocytes. Each square represents peak number of SIV DNA copies during the course of infection from individual macaques with SIVE (red) and macaques without encephalitis (blue). b: The number of SIV DNA copies was assessed in CD14⁺ blood monocytes isolated by magnetic bead separation every 2 weeks after infection from macaques with SIVE (red) and macaques without encephalitis (blue). There was a peak in SIV DNA in CD14⁺ monocytes in two of the three macaques with SIVE from 2 to 8 weeks after infection and one of five macaques without encephalitis at 2 and 10 weeks after infection. Regardless of the development of encephalitis, all but one macaque (no. 145) had detectable SIV DNA in CD14⁺ blood monocytes at least one time during infection. c: Peak SIV p27 production of MDMs (adherent PBMCs) cultured ex vivo. Each square represents peak p27 production of MDMs during the course of infection from individual macaques with SIVE (red) and macaques without encephalitis (blue). d: During the course of infection, p27 production of MDMs (adherent PBMCs) cultured ex vivo for 14 days showed that the three macaques with SIVE (red) produce more p27 in culture than the five macaques without encephalitis (blue). This graph shows p27 values from MDMs of each macaque every 2 weeks after infection. SIV p27 production peaked in macaques that developed SIVE for one or two consecutive time points measured between 4 to 8 weeks after infection. Average p27 production in MDMs from macaques that developed encephalitis was significantly higher than macaques without encephalitis at 4 weeks after infection, *P < 0.05. Because macaques 152 and 154 were experimentally sacrificed, they were not included in statistical analysis. e: Representative image of MDM cultures that were assayed for SIV p27 production by enzyme-linked immunosorbent assay. The culture was immunostained for CD68 (top left, blue, Cy5), SIVgp110 (top right, red, Cy3), and CD3 (bottom left, green, Alexa 488) and visualized by triple-label immunofluorescent laser confocal microscopy. An overlay of the three preceding images is shown in the bottom right. Purple shows co-localization of MDMs and SIVgp110. f: Peak SIV p27 production of nonadherent PBMCs cultured ex vivo. Each square represents peak p27 production of nonadherent PBMCs during the course of infection from individual macaques with SIVE (red) and macaques without encephalitis (blue). g: Longitudinal p27 production of nonadherent PBMCs cultured ex vivo for 14 days shows that two of three macaques with SIVE (red) produce more p27 in culture than the five macaques without encephalitis (blue) at week 2 after infection. Because of unavailable PBMC samples, statistical analyses could not be completed for week 2 after infection. Because macaques 152 and 154 were experimentally sacrificed, they were not included in statistical analysis. Scale bar, 50 μm.

Necropsy survey of the number of infected macrophages and T lymphocytes observed in peripheral organs from eight CD8-depleted rhesus macaques infected with SIV/DeltaB670. Based on histological findings, macaques were retrospectively classified at postmortem for presence of SIV encephalitis. Each organ was immunostained for both CD3/SIVgp110 and CD68/SIVgp110 and visualized by immunofluorescent confocal microscopy. Three observers enumerated the number of infected macrophages (CD68⁺/SIV⁺ cells), infected T cells (CD3⁺/SIV⁺cells), and SIV-infected cells that did not co-label with CD68 or CD3 (CD3⁻/SIV⁺ and CD68⁻/SIV⁺ cells). The black bars represent the mean number of infected cells enumerated for each group (each dot represents the enumeration from an individual field). Macaques with SIVE show more SIV-infected cells in the small bowel and spinal cord but less SIV-infected cells in the lung and thymus than macaques without encephalitis. Many organs have SIV-infected cells that do not co-label with CD3 or CD68. Macrophages are the most common SIV-infected cells in these organs. a: Liver. Median number of infected T cells and macrophages per field in the liver was similar in macaques with and without encephalitis. b: Lung. The median number of infected macrophages and infected cells that did not label with CD68 or CD3 was higher in macaques without encephalitis compared to macaques with SIVE. c: Small bowel. Median number of infected cells that did not label with CD3 or CD68 was statistically significantly higher in macaques with SIVE compared to macaques without encephalitis. d: Spinal cord. Median number of infected macrophages and infected cells that did not label with CD3 or CD68 was statistically significantly higher in macaques with SIVE compared to macaques without encephalitis. e: Spleen. Median number of infected T cells and macrophages per field in the spleen was similar in macaques with and without encephalitis. f: Thymus. The median number of infected macrophages and infected cells that did not label with CD3 was statistically significantly higher in macaques without encephalitis compared to macaques with SIVE. *P < 0.05, **P < 0.01, ***P < 0.001.