Fig 1.
Histopathology in the visceral organs of EEEV infected cynomolgus macaques.
The tissues were collected at the time of euthanasia. Hematoxylin and eosin (H&E) staining was performed on the tissues of all four NHPs. Bar = 200 um.
Fig 2.
Pathology in the amygdala and the hippocampus of EEEV infected cynomolgus macaques.
The tissues were collected at the time of euthanasia. Hematoxylin and eosin (H&E) staining was performed to visualize histopathology. The presence of EEEV RNA and proteins was visualized via in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. H&E, ISH, and IHC were performed on the tissues of all four NHPs. Bar = 100 um (H&E and IHC). Bar = 50 um (ISH). Arrow key: neutrophilic infiltration/neutrophils (black), degenerative/necrotic neurons (red), and vacuolation of the neuropil (green).
Fig 3.
Pathology in the hypothalamus of EEEV infected cynomolgus macaques.
The tissue was collected at the time of euthanasia. Hematoxylin and eosin (H&E) staining was performed to visualize histopathology. The presence of EEEV RNA and proteins was visualized via in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. H&E, ISH, and IHC were performed on the tissues of all four NHPs. Bar = 100 um (H&E and IHC). Bar = 50 um (ISH).
Fig 4.
Histopathology in various parts of the brain tissues of EEEV infected cynomolgus macaques.
The tissues were collected at the time of euthanasia. Hematoxylin and eosin (H&E) staining was performed to visualize histopathology. H&E was performed on the tissues of all four NHPs. Bar = 100 um. Arrow key: degenerative to necrotic neurons (red), vacuolation of the neuropil (green), and microhemorrhage (blue).
Fig 5.
The presence of EEEV RNA in various parts of the brain tissues of infected cynomolgus macaques.
The tissues were collected at the time of euthanasia. The presence of viral RNA was visualized via in situ hybridization (ISH). ISH was performed on the tissues of all four NHPs. Bar = 50 um.
Fig 6.
The presence of EEEV proteins in various parts of the brain tissues of infected cynomolgus macaques.
The tissues were collected at the time of euthanasia. The presence of viral proteins was visualized via immunohistochemistry (IHC). IHC was performed on the tissues of all four NHPs. Bar = 100 um.
Fig 7.
Histopathology in various parts of the spinal cord of EEEV infected cynomolgus macaques.
The tissues were collected at the time of euthanasia. Hematoxylin and eosin (H&E) staining was performed to visualize histopathology. H&E was performed on the tissues of all four NHPs. Bar = 100 um. Arrow key: neutrophilic infiltration/neutrophils (black).
Fig 8.
The presence of EEEV RNA in various parts of the spinal cord of infected cynomolgus macaques.
The tissues were collected at the time of euthanasia. The presence of viral RNA was visualized via in situ hybridization (ISH). ISH was performed on the tissues of all four NHPs. Bar = 50 um.
Fig 9.
The presence of EEEV proteins in various parts of the spinal cord of infected cynomolgus macaques.
The tissues were collected at the time of euthanasia. The presence of viral proteins was visualized via immunohistochemistry (IHC). IHC was performed on the tissues of all four NHPs. Bar = 100 um.
Fig 10.
The presence of EEEV RNA in the astrocytes of infected cynomolgus macaques.
Sections from the thalamus of each NHP were visualized via immunofluorescence assay. Sections were stained for GFAP (green), EEEV (red), and DAPI (blue).
Fig 11.
The presence of EEEV RNA in the microglia of infected cynomolgus macaques.
Sections from the thalamus of each NHP were visualized via immunofluorescence assay. Sections were stained for CD68 (green), EEEV (red), and DAPI (blue).
Fig 12.
The presence of EEEV RNA in the neurons of infected cynomolgus macaques.
Sections from the thalamus of each NHP were visualized via immunofluorescence assay. Sections were stained for NeuN (green), EEEV (red), and DAPI (blue).
Fig 13.
The extracellular distribution of EEEV virions in the thalamus of infected cynomolgus macaques.
Sections from each NHP were examined and representative micrographs from each NHP are shown. Red arrows indicate virus particles. Bar = 200 nm.
Fig 14.
The size of extracellular EEEV virions via transmission electron microscopy (TEM).
Sections from the thalamus of each NHP were examined and representative micrographs from NHPs are shown. Red arrow indicates virus particles.
Fig 15.
The localization of EEEV virions around the myelin sheath of neurons via transmission electron microscopy (TEM).
Sections from the thalamus of each NHP were examined and representative micrographs from each NHP are shown. Red arrows indicate virus particles.
Fig 16.
The localization of EEEV virions within the axons of neurons via transmission electron microscopy (TEM).
Sections from the thalamus of each NHP were examined and representative micrographs of each NHP are shown. Red arrows highlight virus particles. Bar = 200 nm.
Fig 17.
The localization of EEEV virions within an axon of a neuron via transmission electron microscopy (TEM).
Sections from the thalamus of NHP #3 were examined. Two sequential sections, ~80 nm apart, of an axon are shown. Red arrows indicate virus particles. Scale bar = 100 nm.
Fig 18.
The detection of cytopathic vacuoles in the cytoplasm of EEEV infected cells via transmission electron microscopy (TEM).
Sections from the thalamus of infected NHPs were examined. Micrographs of NHP #4 are shown. Scale bars: Panels A and B = 600 nm, C and D = 400 nm, E, F and G = 200 nm, and H = 100 nm.
Fig 19.
The detection of cytopathic vacuoles, nucleocapsid, and budding virions in EEEV infected cells via transmission electron microscopy (TEM).
Blue and red arrows indicate nucleocapsid and virus particles, respectively. Sections from the thalamus of infected NHP #3 were examined. Scale bars: A = 200 nm, B = 100 nm.