C-type lectin receptor DCIR contributes to hippocampal injury in acute neurotropic virus infection

Neurotropic viruses target the brain and contribute to neurologic diseases. C-type lectin receptors (CLRs) are pattern recognition receptors that recognize carbohydrate structures on endogenous molecules and pathogens. The myeloid CLR dendritic cell immunoreceptor (DCIR) is expressed by antigen presenting cells and mediates inhibitory intracellular signalling. To investigate the effect of DCIR on neurotropic virus infection, mice were infected experimentally with Theiler’s murine encephalomyelitis virus (TMEV). Brain tissue of TMEV-infected C57BL/6 mice and DCIR−/− mice were analysed by histology, immunohistochemistry and RT-qPCR, and spleen tissue by flow cytometry. To determine the impact of DCIR deficiency on T cell responses upon TMEV infection in vitro, antigen presentation assays were utilised. Genetic DCIR ablation in C57BL/6 mice was associated with an ameliorated hippocampal integrity together with reduced cerebral cytokine responses and reduced TMEV loads in the brain. Additionally, absence of DCIR favoured increased peripheral cytotoxic CD8+ T cell responses following TMEV infection. Co-culture experiments revealed that DCIR deficiency enhances the activation of antigen-specific CD8+ T cells by virus-exposed dendritic cells (DCs), indicated by increased release of interleukin-2 and interferon-γ. Results suggest that DCIR deficiency has a supportive influence on antiviral immune mechanisms, facilitating virus control in the brain and ameliorates neuropathology during acute neurotropic virus infection.

Whole-brain fluorescence-MRI coregistration for precise anatomical mapping of virus infection

Neurotropic virus infections cause tremendous disease burden. Methods visualizing infection in the whole brain remain unavailable which greatly impedes understanding of viral neurotropism and pathogenesis. We devised an approach to visualize the distribution of neurotropic virus infection in whole mouse brain ex vivo. Optical projection tomography (OPT) signal was coregistered with a unique magnetic resonance imaging (MRI) brain template, enabling precise anatomical mapping of viral distribution, and the effect of type I interferon on distribution of infection was analyzed. Guided by OPT-MR, we show that Langat virus specifically targets sensory brain systems and the lack of type I interferon response results in an anatomical shift in infection patterns in the brain. We confirm this regional tropism, observed with whole brain OPT-MRI, by confocal and electron microscopy to provide unprecedented insight into viral neurotropism. This approach can be applied to any fluorescently labeled target in the brain.

CARD9 Deficiency Increases Hippocampal Injury Following Acute Neurotropic Picornavirus Infection but Does Not Affect Pathogen Elimination

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9−/− and corresponding C57BL/6 wild-type control mice were infected with Theiler’s murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9−/− mice showed an increased loss of neuronal nuclear protein+ mature neurons and doublecortin+ neuronal precursor cells and an increase in β-amyloid precursor protein+ damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8+ T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.

A spatial and cellular distribution of neurotropic virus infection in the mouse brain revealed by fMOST and single cell RNA-seq

Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both human and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques. In this study, we explore the use of fluorescence micro-optical sectioning tomography (fMOST) and single cell RNA sequencing (scRNA-seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of recombinant RABV expressing enhanced green fluorescent protein (EGFP) under different infection routes, and a three-dimensional view of the distribution of RABV in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA-seq. Our fMOST data provide the first evidence that RABV can infect multiple nuclei related to fear independent of different infection routes. More surprisingly, scRNA-seq data indicate that besides neurons RABV can infect macrophages and NK cells in vivo. Collectively, this study draws a comprehensively spatial and cellular map of RABV infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of neurotropic viruses.

Immune response and blood–brain barrier dysfunction during viral neuroinvasion

The blood-brain barrier (BBB), which protects the CNS from pathogens, is composed of specialized brain microvascular endothelial cells (BMECs) joined by tight junctions and ensheathed by pericytes and astrocyte endfeet. The stability of the BBB structure and function is of great significance for the maintenance of brain homeostasis. When a neurotropic virus invades the CNS via a hematogenous or non-hematogenous route, it may cause structural and functional disorders of the BBB, and also activate the BBB anti-inflammatory or pro-inflammatory innate immune response. This article focuses on the structural and functional changes that occur in the three main components of the BBB (endothelial cells, astrocytes, and pericytes) in response to infection with neurotropic viruses transmitted by hematogenous routes, and also briefly describes the supportive effect of three cells on the BBB under normal physiological conditions. For example, all three types of cells express several PRRs, which can quickly sense the virus and make corresponding immune responses. The pro-inflammatory immune response will exacerbate the destruction of the BBB, while the anti-inflammatory immune response, based on type I IFN, consolidates the stability of the BBB. Exploring the details of the interaction between the host and the pathogen at the BBB during neurotropic virus infection will help to propose new treatments for viral encephalitis. Enhancing the defense function of the BBB, maintaining the integrity of the BBB, and suppressing the pro-inflammatory immune response of the BBB provide more ideas for limiting the neuroinvasion of neurotropic viruses. In the future, these new treatments are expected to cooperate with traditional antiviral methods to improve the therapeutic effect of viral encephalitis.

Interferon-Inducible GTPase 1 Impedes the Dimerization of Rabies Virus Phosphoprotein and Restricts Viral Replication

ABSTRACT Rabies, caused by rabies virus (RABV), is an ancient zoonosis and still a major public health problem for humans, especially in developing countries. RABV can be recognized by specific innate recognition receptors, resulting in the production of hundreds of interferon-stimulated genes (ISGs), which can inhibit viral replication at different stages. Interferon-inducible GTPase 1 (IIGP1) is a mouse-specific ISG and belongs to the immunity-related GTPases (IRGs) family. IIGP is reported to constrain intracellular parasite infection by disrupting the parasitophorous vacuole membrane. However, the role of IIGP1 in restricting viral replication has not been reported. In this present study, we found that IIGP1 was upregulated in cells and mouse brains upon RABV infection. Overexpression of IIGP1 limited RABV replication in cell lines and reduced viral pathogenicity in a mouse model. Consistently, deficiency of IIGP1 enhanced RABV replication in different parts of mouse brains. Furthermore, we found that IIGP1 could interact with RABV phosphoprotein (P protein). Mutation and immunoprecipitation analyses revealed that the Y128 site of P protein is critical for its interaction with IIGP1. Further study demonstrated that this interaction impeded the dimerization of P protein and thus suppressed RABV replication. Collectively, our findings for the first reveal a novel role of IIGP1 in restricting a typical neurotropic virus, RABV, which will provide fresh insight into the function of this mouse-specific ISG. IMPORTANCE Interferon and its downstream products, ISGs, are essential in defending against pathogen invasion. One of the ISGs, IIGP1, has been found to constrain intracellular parasite infection by disrupting their vacuole membranes. However, the role of IIGP1 in limiting viral infection is unclear. In this study, we show that infection with a typical neurotropic virus, RABV, can induce upregulation of IIGP1, which, in turn, suppresses RABV by interacting with its phosphoprotein (P protein) and thus blocking the dimerization of P protein. Our study provides the first evidence that IIGP1 functions in limiting viral infection and provides a basis for comprehensive understanding of this important ISG.