Fig 1.
HAstV-NIH infects neurons with the highest burden in the cerebellum and brainstem.
(A—H) Astrovirus capsid protein (brown) in neuronal perikarya and projections (dendrites and axons) in indicated brain structures. Heatmap (yellow-low to red-high) of the neuronal virus infection burden along the neural axis is shown on the left of each image panel. Colors are based on the normalized counts of AstV+ neurons per mm2 of tissue area for all structures, except for Purkinje cells (PCs) in the cerebellar cortex, which is based on the number of AstV+ PC profiles per linear mm of PC layer. (I) Radar graph represents the entire brain clockwise from the cerebral cortex to the medulla and shows the normalized numbers of AstV+ neurons in each structure (data are presented as mean counts [red line] ± SE [gray dashed lines]). (J and K) Double immunofluorescent staining for the neuronal somatodendritic marker MAP2 (red), AstV capsid protein (green), and DAPI nuclear counterstain (blue) in the indicated brainstem structures with high neuronal infection burden. Note: (i) accumulation of aging pigment lipofuscin, which is known to be autofluorescent, produces an intense red signal in the perikarya of AstV-negative (AstV-) MAP2++ neurons and dendritic profiles and this signal colocalizes (yellow) with AstV (green) signal in infected neurons; and (ii) there is a paucity of AstV-/MAP2++ dendritic profiles and MAP2 signals are either low (MAP2+) or absent (MAP2-) in AstV+ dendritic profiles. Labeling keys used in (J and K) are provided at the bottom of the figure. Scale bars: 10 μm.
Fig 2.
Functional genomic analysis of changes in transcriptional regulation of neurophysiology in AstV-ND.
(A) Coordinated shifts in transcriptional regulation of brain homeostasis in AstV-ND-1-NIH compared to a normal age-matched control. Plotted are negative log10 FDR-adjusted p-values (-log10 p-adj) for major Gene Ontology (GO) terms of interest determined by SET. Dashed line indicates the significance cut-off. Inferred changes in AstV-ND-1-NIH compared to normal control for each term are indicated in the gray box. (B) Multi-source functional enrichment for downregulated gene expression in AstV-ND. The plot shows significantly enriched terms of interest across multiple ontologies (x-axis) with their p-adj values (left y-axis) and the number of downregulated genes annotated to each term (right y-axis). Genomic ontology sources and their corresponding terms are indicated by the same color.
Table 1.
Analysis of disruption of neurophysiology in AstV-ND at the levels of transcriptional regulation, protein expression, and cell morphology/topology/function.
Fig 3.
AstV replication in neurons triggers loss of their afferent innervation.
(A—H) Representative images show a side-by-side comparison of the density of synaptophysin-immunoreactive presynaptic terminals in the neuropil surrounding uninfected neurons (“Normal age-matched control” column) versus astrovirus-infected neurons (“AstV-ND-1” column) in indicated brain regions. Each panel is composed of (1) an autofluorescence-simulated image that was pseudo-colored as hematoxylin-eosin (H&E) staining to serve as a topographical reference and to aid identification of intra- and extra-neuronal autofluorescent lipofuscin granules in corresponding immunofluorescent images (see Materials and Methods) and (2) immunoreactivity signals for the pan-synaptic marker synaptophysin (red), astrovirus (green), and DAPI nuclear counterstain (blue) in the corresponding tissue field. Outlines of the select neuronal perikarya/dendrites correspond to the same neurons shown in the H&E reference images and corresponding immunofluorescent images. Labeling keys used in (A—H) are provided at the bottom of the figure. Scale bars: 10 μm.
Fig 4.
AstV infection is associated with disruption of both excitatory and inhibitory neurotransmission.
(A—D) Representative images show a side-by-side comparison of the density (brown immunoreactivity) of presynaptic compartments of excitatory glutamatergic (vesicular glutamate transporter 1 [VGLUT1]+) synapses (A and B) and inhibitory GABA-ergic (vesicular GABA transporter [VGAT]+) synapses (C and D) in the brainstem of a normal age-matched control subject (A and C) and in the brainstem of AstV-ND-1-NIH (B and D). Note an extensive loss of the excitatory (B) and inhibitory (D) presynaptic puncta in AstV-ND-1-NIH, compared to a normal age-matched control (A and C, respectively). Scale bars: 10 μm.
Fig 5.
AstV infection is associated with disruption of structural integrity of neuronal somatodendritic compartments.
(A—D) Representative images show a side-by-side comparison of the MAP2 immunoreactivity (brown) in somatodendritic compartments of neurons in the pontine nuclei (A and B) and medullary reticular formation (C and D) in the brainstem of a normal age-matched control subject (A and C) and in the brainstem of AstV-ND-1-NIH (B and D). Note an extensive loss of the MAP2 immunoreactivity in AstV-ND-1-NIH, compared to a normal age-matched control. Scale bars: 10 μm.
Fig 6.
Functional genomic analysis of transcriptional regulation of immune responses in the brain of patients with AstV-ND.
(A) Coordinated shifts in transcriptional regulation of immune processes in the brain of AstV-ND-1-NIH compared to a normal age-matched control. Plotted are negative log10 FDR-adjusted p-values (-log10 p-adj) for major Gene Ontology (GO) terms of interest determined by SET. Dashed line indicates the significance cut-off. Inferred changes in AstV-ND-1-NIH compared to normal control for each term are indicated in the gray box. (B) Multi-source functional enrichment for upregulated gene expression in AstV-ND. The plot shows significantly enriched terms of interest across multiple ontologies (x-axis) with their p-adj values (left y-axis) and the number of upregulated genes annotated to each term (right y-axis). Genomic ontology sources and their corresponding terms are indicated by the same color.
Fig 7.
Cellular responses to AstV infection in the brainstem of an immunocompromised adult.
(A—H) Representative images show perivascular (A, C, E, G, and H) and perineuronal (B, D, and F) tissue in adjacent sections of the medulla that illustrate: (1) perivascular cellular infiltrate (A) and perineuronal hypercellularity (B) (hematoxylin and eosin [H&E]); (2) moderate astrocytosis (C and D; glial fibrillary acidic protein [GFAP]; brown) with (i) hypertrophy of astrocytic somata and perivascular end foot processes (C) and (ii) retraction of perineuronal astrocytic processes and a cell with microglial morphology (putative microglia) that is in close apposition to the neuronal membrane (D); (3) CD68+ perivascular/parenchymal border macrophages and intraparenchymal microglia/macrophages (E; brown); (4) intraparenchymal/perineuronal phagocytic microglia/macrophages (F; brown); (5) infiltrating (extravasated/perivascular/intraparenchymal) CD3+ T cells (G; brown); and (6) absence of infiltration by CD20+ B cells (H). Scale bars: 10 μm.
Table 2.
Analysis of immune responses associated with AstV-ND at the levels of transcriptional regulation, protein expression, and cell morphology/topology/function.