Table 1.
Summary of CNS region names, structures/types of neurons, reference virus titers, extent/intensity, timing of neuronal WNV-labeling, and references to representative images of WNV-labeled neurons.
Fig 1.
WNV-labeled corticospinal motor neurons in the cortical layer 5 of the primary motor cortex.
Inset shows one of the circled areas at higher magnification. Red arrowhead points to a large WNV-labeled corticospinal motor neuron (Betz cell). Note that all three circled WNV- labeled Betz cells are damaged and undergoing neuronophagia (7 dpi). Also note an adjacent long axonal profile that contains WNV-antigens (red arrows). Scale bars: 100 μm.
Fig 2.
WNV-labeled neurons in the subcortical structures, midbrain, pons, and medulla oblongata.
(A) Neurons in the motor thalamus display intense WNV-labeling in the neuronal perikarya and dendritic/axonal profiles (7 dpi). (B) WNV-labeled neuron in the basal ganglia. Note its close vicinity to a perivascular inflammatory infiltrate (7 dpi). (C) WNV-labeled neurons in the substantia nigra pars compacta (9 dpi). (D) Red nucleus magnocellular part (9 dpi). Perikarya of large neurons display granular WNV-labeling. Also note WNV-labeled neuritic profile emanating from the infected neuron in the lower-left corner of the field. Representative images of WNV-labeled neurons in the brainstem are shown in E (pontine nuclei; 7 dpi), F (vestibular nuclei; 9 dpi), G (medullary reticular formation; 7 dpi), H (inferior olivary nuclei; 7 dpi), and I (accessory cuneate nucleus; 9 dpi). Scale bars: 100 μm.
Fig 3.
WNV-labeled neurons in the deep cerebellar nuclei.
(A) Low magnification overview image showing many WNV-labeled neurons in the dentate nucleus (Dt), emboliform nucleus (Emb), and globose nucleus (Glo) (9 dpi). B and C show corresponding boxed areas at higher magnification. Scale bars: 100 μm.
Fig 4.
WNV-labeled neurons in the cerebellar cortex.
Original views of WNV-labeled neurons at 7 dpi (A) and 9 dpi (C). B and D show the corresponding markup images of A and C after applied “WNV-labeled cell segmentation” image analysis algorithm (WNV immunoreactivity: red, strong; orange, moderate; yellow, weak; negative, blue). Note an increasing WNV immunoreactivity in the somatodendritic compartments of Purkinje cells from 7 dpi to 9 dpi (compare B and D) and variable immunoreactivity in small groups of granule neurons only at 9 dpi (D). M, molecular layer; P, Purkinje cell layer; G, granule cell layer. Scale bars: 100 μm.
Fig 5.
WNV-labeled neurons in the spinal cord.
Representative images of WNV-labeled neurons in the cervical (A and D), thoracic (B and E), and lumbar regions (C and F) of the spinal cord are shown on indicated dpi. Approximate boundaries of the spinal cord gray matter are outlined in the overview images. Clarke’s column is shown by magenta overlay in E. Round insets show the corresponding circled areas at higher magnification. Note that the majority of WNV-labeled neurons occupy a medial portion of the Clarke’s column. Vh, ventral horns; CC, Clarke’s column. Bars in overview images: 1000 μm. Bars in the round insets: 100 μm.
Table 2.
Summary of neurological functions related to the structures containing WNV-labeled neurons.
Fig 6.
Ultrastructural localization of WNV particles in the cerebellar cortex.
Shown are electron microscopy images of dendritic arbors of Purkinje cells (PCs). Some asymmetric axo-dendritic synapses are shown by green overlays. In panels (A and D): white arrows point to the postsynaptic density (PSD) and black arrows show synaptic clefts (SC). In some panels the groups of virions are outlined by yellow dashed circles/ovals. Insets in (A, B, E, and F) show the corresponding red boxed areas at higher magnification. (A) Bifurcating PC dendrite contains multiple spherical electron-dense virions. Inset shows several virions in close vicinity to the dendritic spine (DS) which is synapsing with axonal terminal (AT). (B) A shaft of another PC dendrite is shown at higher magnification. Virions can be seen in association with the hypolemmal cisternae of the agranular endoplasmic reticulum (AER; white arrows) and microtubule structures (white arrowheads). Inset shows three virions adjacent to the AER (white arrow) and one virion inside the vesicle adjacent to microtubules covered with a “cottony” material. (C and D) Virions within PC dendrites are found in close vicinity to synapses. (E) Virion-containing vesicles are in the presynaptic position in the axonal terminal. Another axonal terminal in the lower right corner contains synaptic vesicles (black arrows) and one large dense-core vesicle (black arrowhead). (F) Virions are enclosed within the autophagosome-like vesicles with double-layered membrane. Inset shows one such vesicle at presynaptic position (~40 nm away from the active zone of the synapse). M, mitochondria. Scale bars: 100 nm.
Fig 7.
WNV particles versus dense core vesicles in the spinal cord.
Shown are electron microscopy images of ventral horns of cervical and lumbar regions of the spinal cord. (A) Four large vesicles containing virions are shown by yellow overlays. These vacuoles lie close to a degenerating myelin (My) sheath of axon. Degenerating part of the myelin sheath is shown by magenta overlay. (B) Higher magnification image of the boxed area in (A) shows several virions of ~ 40 nm in diameter. Note a lighter lipid layer underneath the darker envelope layer especially clearly visible in one of the virions (arrow). (C and D) Compare two axonal areas containing virions (yellow overlay in C) or dense core vesicles (D). Insets show red circled areas at higher magnification. Approximate diameters of virions and vesicles are indicated. Scale bars: 100 nm (additional scale bars for dense core vesicles are indicated within the inset in D).
Fig 8.
Proposed directionality of WNV spread based on the neuroanatomical connectivity and time of immunohistochemical virus detection.
The connectograms illustrate most probable routes and directionality of WNV spread within the CNS in our NHP model of neuroinfection at 7 dpi (A) and 9/10 dpi (B). Construction and elements of the connectogram are described in Materials and Methods and text. Each arrow has the same color as the structure in the ring from which it originates. The circled numbers 1 to 4 represent most probable “order” of infected neurons within the corresponding anatomical structures. Solid arrows indicate most probable routes of anterograde spread; dashed arrows indicate most probable routes of retrograde spread. White lines indicate the possibility of both, anterograde and retrograde virus spread, due to existence of reciprocal connections between the same orders of neurons. Abbreviations: Mthal, motor thalamus; BG, basal ganglia; CSMN, corticospinal motor neurons; SNC, substantia nigra pars compacta; RnM, red nucleus magnocellular; SMN, spinal motor neurons (C—cervical; T—thoracic; L—lumbar); CC, Clarke’s column; DCN, deep cerebellar nuclei; ACu, Accessory cuneate nucleus; IO, inferior olivary nuclear complex; MeRF, Medullary reticular formation; Ve, vestibular nuclei; PN, pontine nuclei.