gProfiler Multiquery Upregulated Genes.

Astroviruses (AstVs) can cause of severe infection of the central nervous system (CNS) in immunocompromised individuals. Here, we identified a human AstV of the VA1 genotype, HAstV-NIH, as the cause of fatal encephalitis in an immunocompromised adult. We investigated the cells targeted by AstV, neurophysiological changes, and host responses by analyzing gene expression, protein expression, and cellular morphology in brain tissue from three cases of AstV neurologic disease (AstV-ND). We demonstrate that neurons are the principal cells targeted by AstV in the brain and that the cerebellum and brainstem have the highest burden of infection. Detection of VA1 AstV in interconnected brain structures such as thalamus, deep cerebellar nuclei, Purkinje cells, and pontine nuclei indicates that AstV may spread between connected neurons transsynaptically. We found transcriptional dysregulation of neural functions and disruption of both excitatory and inhibitory synaptic innervation of infected neurons. Importantly, transcriptional dysregulation of neural functions occurred in fatal cases, but not in a patient that survived AstV-ND. We show that the innate, but not adaptive immune response was transcriptionally driving host defense in the brain of immunocompromised patients with AstV-ND. Both transcriptome and molecular pathology studies showed that most of the cellular changes were associated with CNS-intrinsic cells involved in phagocytosis and injury repair (microglia, perivascular/parenchymal border macrophages, and astrocytes), but not CNS-extrinsic cells (T and B cells), suggesting an imbalance of innate and adaptive immune responses to AstV infection in the brain as a result of the underlying immunodeficiencies. These results show that VA1 AstV infection of the brain in immunocompromised humans is associated with imbalanced host defense responses, disruption of neuronal somatodendritic compartments and synapses and increased phagocytic cellular activity. Improved understanding of the response to viral infections of the human CNS may provide clues for how to manipulate these processes to improve outcomes.</div

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.</p

Maximum-likelihood phylogeny of the mammal and avian astroviruses.

The circular dendrogram is constructed from the ORF2/capsid precursor protein sequences of 229 astroviruses retrieved from the NCBI protein sequence database (see Materials and Methods for details). Thick branches were significantly supported (bootstrap percentage > 0.70) by the data. Mammalian and avian lineages are labeled at their most basal branches. The color labeling (indicated in the top right corner) is assigned based on the type of samples in which the virus was detected. Three astroviruses belonging to the Human VA1 clade that are associated with this study are indicated by bold red color. Other clades of human astroviruses are indicated in bold black color on the periphery (right side) of the dendrogram. Human astroviruses associated with CNS infections are indicated by black dots. (PDF)</p

Analysis of disruption of neurophysiology in AstV-ND at the levels of transcriptional regulation, protein expression, and cell morphology/topology/function.

Analysis of disruption of neurophysiology in AstV-ND at the levels of transcriptional regulation, protein expression, and cell morphology/topology/function.</p

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.</p

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.</p

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.</p

Detection of astrovirus in the brain of an immunocompromised patient with an encephalitis.

(A) Fold change relative to control for probes to virus families hybridizing to RNA from the brain of the patient with AstV-NIH. (B) Detection of the astrovirus RNA in the brain of the patient with AstV-NIH by PCR. (C and D) In situ hybridization signals (magenta-red) in indicated brain tissue samples using the negative strand astrovirus RNA probe (upper panels) or RNA probes for actin as controls (lower panels). (PDF)</p

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.</p

Reported premorbid conditions and neuropathology associated with AstV-ND in humans and animals.

Reported premorbid conditions and neuropathology associated with AstV-ND in humans and animals.</p