Astrocyte Infection during Rabies Encephalitis Depends on the Virus Strain and Infection Route as Demonstrated by Novel Quantitative 3D Analysis of Cell Tropism

Abstract

Although conventional immunohistochemistry for neurotropic Rabies virus (RABV) usually shows a high preference for neurons, non-neuronal cells are also potential target cells and abortive infection of astrocytes is considered a main trigger of innate immunity in the CNS. While in vitro studies indicated differences between field and less virulent lab-adapted RABVs, a systematic and quantitative comparison of astrocyte tropism in vivo is lacking. Here, a recently developed solvent-based tissue clearing technique was used to measure the RABV cell tropism in infected brains. Immunofluorescence analysis of 1 mm-thick tissue slices enabled 3D segmentation and quantification of infection frequencies of astrocytes and neurons. Comparison of highly virulent street virus clones from fox, dog, and raccoon with three lab strains of intermediate and low virulence revealed remarkable differences in the ability to infect astrocytes in vivo. While all viruses and infection routes led to comparable neuron infection frequencies, striking differences were detected for the infection of astrocytes. Consistent and inoculation route-independent astrocyte infection by field viruses, together with route-dependent or undetectable astrocyte infection by lab-adapted or vaccine viruses strongly suggests a model in which the ability to establish productive astrocyte infection in vivo functionally distinguishes field and attenuated lab RABV strains.

Light Sheet Microscopy-Assisted 3D Analysis of SARS-CoV-2 Infection in the Respiratory Tract of the Ferret Model

“…Additionally, viral antigen has been detected in the intestine of ferrets [ 31 ], hamsters [ 34 , 35 ], and rhesus macaques [ 44 ]. Previous studies with other viral pathogens demonstrated that volumetric 3D imaging using TOC and LSFM is a highly valuable tool to assess the comprehensive distribution of virus infection in vivo [ 51 , 54 ]. Additional immunostaining against tissue-specific cell markers may further facilitate the investigation of the global SARS-CoV-2 cell tropism in affected tissues.…”

“…This eliminates the need for physical sectioning and allows acquisition of intact three-dimensional (3D) structures using only optical sectioning, e.g., in light sheet fluorescence microscopy (LSFM) [ 50 ]. Lately, the opportunities and advantages of TOC for virus research have been demonstrated in several studies [ 51 , 52 , 53 , 54 , 55 , 56 , 57 ]. While two approaches to 3D imaging of SARS-CoV-2-infected lung tissue have been described recently [ 58 , 59 ], neither of them is capable of direct visualization of SARS-CoV-2 infection via virus-specific antigen staining.…”

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

“…Emerging evidences have shown that RABV is not a strict neurotropic virus and besides neurons it can infect other cell types 17, 18 . To comprehensively investigate the infection susceptibility of diverse cell populations and their contributions to RABV pathogenesis, we performed droplet-based scRNA-seq (10×Genomics) on a total of six mouse brain samples including two from uninfected mice (healthy: n=2), two from mice with paralysis (paralyzed: n=2) and two from mice in the moribund stage post RABV infection (moribund: n=2) (Figure 4A).…”

Cell-type specific innate immune responses shape rabies virus tropism

“…Whereas most of the research is focussing on the discovery of RABV receptors (1)(2)(3)(4) and their interaction with the RABV G-protein (21), less research focusses on how cellular host immune responses shape RABV tropism and how the type of response of the different neural cell types present in the CNS could collectively lead to the establishment of an antiviral response. Recently, several publications reported infection of different glial cells in vivo, particularly astrocytes (27,28) and Schwann cells (9), depending on the viral strain and the infection route used (8). Still, we are far from understanding the molecular pathways underlying susceptibility to RABV infection although it is crucial in determining infection outcome.…”

“…Several receptors are known to accelerate RABV entry to the cell (1–4) and yet, none of them seems to be essential for successful infection (5,6) questioning a marked cellular tropism. Although RABV is classically reported as strictly neurotropic (7), recent studies report profound infection of glial cells in vivo depending on the viral strain and the infection route used (8,9). Here, we provide evidence that human neural stem cell (hNSC)-derived neurons (hiNeurons) and astrocytes (hiAstrocytes) are highly susceptible towards infection with the virulent field RABV strain Tha and less virulent Th2P-4M.…”