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Pathogenesis and outcome of VA1 astrovirus infection in the human brain are defined by disruption of neural functions and imbalanced host immune responses [1]

['Olga A. Maximova', 'Laboratory Of Infectious Diseases', 'National Institute Of Allergy', 'Infectious Diseases', 'National Institutes Of Health', 'Bethesda', 'Maryland', 'United States Of America', 'Melodie L. Weller', 'Secretory Physiology Section']

Date: 2023-08

( 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 mm 2 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.

Together, these findings demonstrate that neurons are the principal cells targeted by HAstV-NIH in the brain and that the cerebellum and brainstem carry the highest burden of infection. This is consistent with most reports that detected AstV in the CNS neurons of infected humans and animals ( S1 Table ). In addition, these findings suggest a transsynaptic mode of virus spread within human CNS with concurrent disruption of integrity of neuronal somatodendritic compartments.

Furthermore, we found AstV-infected neurons in several specific brain structures. The same structures have been reported to harbor neurons infected by another neuropathogenic virus, West Nile virus, that spreads within the CNS transsynaptically [ 35 ]. These AstV-infected structures were the basal ganglia (i.e., putamen; Fig 1B ), thalamus ( Fig 1D ), deep cerebellar nuclei ( Fig 1E ), Purkinje cells in the cerebellar cortex ( Fig 1F ), pontine nuclei ( Fig 1G and 1J ), and medullary reticular formation ( Fig 1H and 1K ). This indicates that AstV may also spread between connected neurons transsynaptically, in both anterograde and retrograde directions.

Next, we used antibody to AstV capsid protein [ 13 , 17 ] and performed immunohistochemistry to identify AstV-infected cells and their distribution within the human brain. The topology and morphology of AstV infected cells ( Fig 1A–1H ), as well as confirmatory double immunofluorescent staining for AstV capsid protein and microtubule associated protein 2 (MAP2; pan-neuronal somatodendritic marker) ( Fig 1J and 1K ), unambiguously demonstrated that HAstV-NIH infects exclusively neurons. Based on the normalized counts of infected neurons (see Materials and Methods ), a caudal gradient in the burden of neuronal virus infection along the neural axis was evident: from a low level infection in the cerebral cortex to a high level in the cerebellum (i.e., deep cerebellar nuclei and Purkinje cells [PCs] in the cerebellar cortex) and brainstem (i.e., pontine nuclei and medullary reticular formation) ( Fig 1A–1I ). AstV capsid protein was present not only in the perikarya of neurons, but also in their projections (dendrites and axons) over a substantial distance from the neuronal cell body. Notably, the areas containing infected neurons also displayed a general paucity in MAP2 immunoreactivity, and somatodendritic compartments that harbored AstV capsid protein accumulations were depleted of MAP2 ( Fig 1J ) (compared to virus negative somatodendritic compartments, Fig 1K ), suggesting disruption of integrity of the neuronal somatodendritic compartments.

Sequencing of the capsid precursor protein gene of HAstV-NIH, followed by phylogenetic analysis ( S2 Fig ), placed this virus within the VA1-HMO (Virginia/Human-Mink-Ovine-like) clade (reviewed in [ 12 ]), together with four other neuropathogenic AstVs: HAstV-PS [ 13 ], HAstV-VA1/HMO-C-PA [ 15 ], HAstV-SG [ 16 ], and HAstV-VA1/HMO-C-UK1(a) [ 17 ]. VA1/HMO-C AstVs, unlike classic astroviruses HAstV 1–8, are neuropathogenic, particularly in immunocompromised patients (reviewed in [ 12 ]).

We investigated the etiology of encephalitis in a 58-year-old-man who had undergone umbilical cord blood transplant for lymphoma and received immunosuppression for graft-versus-host disease (see Materials and Methods for the case report). RNA was isolated from a frozen portion of the patient’s brain and hybridized to a virus chip containing over 3000 probes for viral families and the predominant virus sequences detected (> 10,000-fold change over normal control) belonged to the family of Astroviridae ( S1A Fig ). We confirmed this finding by detecting AstV sequences by PCR in another portion of the patient’s brain tissue, but not in a normal control human brain ( S1B Fig ), and by finding AstV RNA using in situ hybridization in the brain tissue from the patient, but not from a normal control brain ( S1C and S1D Fig ). The virus was designated as HAstV-NIH (Genbank accession number OP293097.2).

Fatal outcome AstV infection in the human brain is defined by disruption of neurophysiology

To understand the effect of AstV infection on the CNS at a molecular level, we performed RNA-seq and interrogated changes in gene expression in the brain of the patient with HAstV-NIH. We first used an agnostic approach that allows analysis of major coordinated shifts in the levels of gene expression from overall cumulative distribution in a particular tissue, without the need to determine the differentially expressed genes (DEGs). For this, we used the Panther statistical enrichment test (SET) [36]. SET uses unnormalized expression values (i.e., number of transcript reads > 0, for this study) for all expressed genes and returns the significantly enriched gene ontology (GO) terms for genes whose expression values were above or below the overall cumulative distribution. We designated genes whose expression levels in normal control brain were above the cumulative distribution as highly expressed and genes whose expression levels were below the cumulative distribution as suppressed. To infer about the functionality of major transcriptional shifts associated with AstV-ND, we performed SET on the unnormalized gene expression data from the brain of the patient with HAstV-NIH and from the brain of an age-matched individual without known neurological disease. For this comparison, we chose to use a normal control sample from the thalamus, which clustered together with the AstV-ND sample based on gene expression data (see Materials and Methods and S3 Fig) and was also age-matched.

Compared to a normal age-matched control, transcriptional modulation of chemical synaptic transmission in the brain of the patient with HAstV-NIH shifted below the significance cut-off value (False Discovery Rate [FDR] adjusted p value; p-adj < 0.05) (Fig 2A), indicating that genes associated with this term were no longer highly expressed. Conversely, the functional GO categories associated with the neuron death and gliogenesis shifted to higher levels of significance in the brain of the AstV-ND-1-NIH patient compared to a normal brain, indicating that these processes were upregulated.

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TIFF original image Download: 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 log 10 FDR-adjusted p-values (-log 10 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. https://doi.org/10.1371/journal.ppat.1011544.g002

We next asked whether the transcriptional dysregulation of neurophysiology was a common feature of human AstV-ND associated with AstVs of the VA1 genotype. Accordingly, we investigated the differential gene expression in the brain samples from three patients with AstV-ND: (i) AstV-ND-1-NIH (infected with HAstV-NIH), (ii) AstV-ND-2-NY (infected with HAstV-PS [13]), and (iii) AstV-ND-3-France (infected with HAstV-VA1/HMO-C-PA [15]). The two outside cases occurred in adolescent males with an underlying primary B cell immunodeficiency (X-linked agammaglobulinemia, XLA); one patient (AstV-ND-2-NY) died and the other (AstV-ND-3-France) survived the infection (S1 Table). Postmortem brain tissue was obtained from the two fatal cases (AstV-ND-1-NIH and AstV-ND-2-NY) and from a biopsy obtained 4.5 years after the onset of neurological symptoms in the patient who survived his infection (Ast-ND-3-France). The disease duration spanned several months to years in these three cases and while the time intervals from the onset of neurological disease to obtaining the brain samples varied, all patients had severe neurological findings when the tissue was obtained. In addition, while our RNA-seq was focused on the human transcripts, we were able to detect astrovirus sequence reads in the brain samples from all three patients with AstV-ND, but not in any of normal control brain samples, and all these astrovirus sequences were mapped to a reference VA1/HMO clade astrovirus (HAstV-VA1/HMO-C-UK1; [3]) (S2 Table). Thus, we consider these three cases of AstV-ND comparable based on the following common criteria: (i) underlying impairment of adaptive immunity (specifically, B cell immunity); (ii) neurological disease caused by AstV of VA1 genotype; and (iii) analysis of the pathogenesis in the brain at the peak of the disease.

The DEGs for each AstV-ND case were determined by comparisons to the appropriate normal controls (S3 Fig), followed by a side-by-side functional genomic analysis using a multiquery feature of gProfiler (see Materials and Methods).

Functional analysis of genes that were downregulated in the brain of three patients with AstV-ND showed that virtually every neuronal subcellular compartment was affected in the two fatal cases (AstV-ND-1-NIH and AstV-ND-2-NY), but not in the brain of the patient that survived the infection (AstV-ND-3-France). These included genes encoding both excitatory and inhibitory synapses (glutamatergic and GABA-ergic, respectively), neuronal cell bodies, dendrites, and axons (Fig 2B; GO Cellular component). This corresponded to functional impairment of the biological processes such as neurotransmission and synapse organization (Fig 2B; Biological process and Reactome). Interestingly, both AstV-ND-1-NIH and AstV-ND-2-NY also had similar significant enrichments in human disease associations, which included Human Phenotype Ontology (HP) terms describing abnormalities in electrophysiology, movement, coordination, and higher mental function (Fig 2B; Human phenotype). The complete results of functional genomic analysis of the downregulated genes in three AstV-ND cases are provided in S1 File (gProfiler Multiquery Downregulated Genes). Taken together, these results indicate that fatal AstV-ND is associated with a severe transcriptional dysregulation of neuronal cellular compartments and impairment of neurotransmission.

Based on the downregulation of genes associated with synapses (Fig 2B and S1 File), we examined formalin fixed paraffin-embedded (FFPE) tissue sections from AstV-ND-1-NIH by immunohistochemistry using antibodies to confirm changes in expression of proteins important for synapse organization and chemical synaptic transmission (excitatory glutamatergic and inhibitory GABA-ergic) (Table 1). Compared to a normal age-matched control, expression of synaptophysin (SYP), a protein localized to synaptic vesicles of presynaptic compartments of all types of synapses, was greatly depleted in the thalamus, midbrain, pons, and medulla of the AstV-ND-1-NIH patient (Fig 3). Importantly, synaptophysin expression was lost in the neuropil surrounding AstV-infected neurons (Fig 3B–3H). This confirms the disruption of synapse organization identified at the gene expression level (Fig 2B and Table 1 and S1 File) and indicates that AstV infection of neurons triggers loss of their afferent innervation.

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TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1011544.g003

Expression of the vesicular glutamate transporter 1 (a protein localized to excitatory glutamatergic synapses) was markedly reduced (Fig 4A and 4B). This confirms the disruption of excitatory synaptic transmission identified at the gene expression level (Fig 2B and Table 1 and S1 File) and indicates that AstV infection of neurons disturbs excitatory afferent innervation of infected neurons. Expression of the vesicular GABA transporter (a protein localized to inhibitory GABA-ergic synapses) was also markedly reduced (Fig 4C and 4D), confirming the transcriptional dysregulation of inhibitory synaptic transmission and inhibitory afferent innervation of infected neurons (Fig 2B and Table 1 and S1 File).

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TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1011544.g004

Since we observed depletion of MAP2 in the somatodendritic profiles that harbored AstV capsid protein by a double fluorescent immunostaining (Fig 1J and 1K) and our functional genomic analysis also revealed downregulation of genes associated with the neuronal somatodendritic compartments (Fig 2B and S1 File), we further examined changes in MAP2 expression at the protein level in the brainstem of AstV-ND-1-NIH patient by immunohistochemistry, in comparison to a normal age-matched control. As anticipated, MAP2 expression was markedly reduced in the neuronal somatodendritic compartments of AstV-ND-1-NIH compared to a normal age-matched control (Fig 5 and Table 1 and S1 File). This confirms that AstV infection of neurons in the brain of AstV-ND-1-NIH disrupted the integrity and function of neuronal somatodendritic compartments.

Taken together, these results validate the gene expression data by demonstrating the loss of expression of critical proteins functioning in the synaptic and somatodendritic compartments of neurons. The net impact of AstV infection of neurons was disruption of synaptic and somatodendritic integrity, loss of afferent innervation of infected neurons, and impairment of both excitatory and inhibitory neurotransmission. Impairment of these essential neural functions may have contributed to the fatal outcome in AstV-ND-1-NIH, and possibly in AstV-ND-2-NY.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011544

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