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 high preference for neurons, non-neuronal cells are also potential targets, and abortive astrocyte infection 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, quantitative comparison of astrocyte tropism in vivo is lacking. Here, solvent-based tissue clearing was used to measure RABV cell tropism in infected brains. Immunofluorescence analysis of 1 mm-thick tissue slices enabled 3D-segmentation and quantification of astrocyte and neuron infection frequencies.Comparison of three highly virulent field virus clones from fox, dog, and raccoon with three lab-adapted strains revealed remarkable differences in the ability to infect astrocytes in vivo. While all viruses and infection routes led to neuron infection frequencies between 7-19%, striking differences appeared for astrocytes. Whereas astrocyte infection by field viruses was detected independent of the inoculation route (8-27%), only one lab-adapted strain infected astrocytes route-dependently [0% after intramuscular (i.m.) and 13% after intracerebral (i.c.) inoculation]. Two lab-adapted vaccine viruses lacked astrocyte infection altogether (0%, i.c. and i.m.). This suggests a model in which the ability to establish productive astrocyte infection in vivo functionally distinguishes field and attenuated lab RABV strains.Cells 2020, 9, 412 2 of 20 M [4-10], and neuronal survival regulation by G [11][12][13][14]. Most pathogenicity studies, however, were performed on already attenuated virus backbones. Thus, differences in their ability to cause disease between highly virulent field virus isolates and lab-adapted, less pathogenic RABV strains are poorly understood. Moreover, it is unclear how molecular differences identified in virulent and attenuated viruses affect virus replication and spread in the infected animal and how the complex virus-host interplay eventually results in either disease or an abortive infection.In vivo, after infection of neurons, RABV spreads trans-synaptically from infected to connected neurons [15]. Retrograde axonal transport of RABV over long distances [16,17] along microtubules [18,19] is a key step in RABV neuroinvasion and is essential for infection of the central nervous system (CNS) through the peripheral nervous system. Co-internalization together with the neuronal p75NTR (tumor necrosis factor receptor superfamily member 16; TNFRSF16) receptor, subsequent retrograde axonal transport of RABV particles in endocytic vesicles, and post-replicative anterograde axonal transport of newly formed RABV have been visualized by live virus particle tracking in sensory neurons [20,21], emphasizing the capacity of hijacking neuron-specific machineries for long distance transport to synaptic membranes. However, internalization and axonal transport of lab-adapted viruses [20,21] together with the use of vaccine virus vectors for trans-syn...

Genetic and Phenotypic Characterization of a Rabies Virus Strain Isolated from a Dog in Tokyo, Japan in the 1940s

“…We therefore hypothesized that infection in this area may be involved in aggressive behaviors that are frequently observed in humans and animals infected with RABV [58,59]. In order to obtain clues for elucidation of the pathogenic mechanisms of rabies, in the near future, we will exhaustively compare distributions of cells infected with the Koma, 1088 and RABV-Dog strains in mouse brains, by using a 3D imaging technique that was reported recently [24,60].…”

3D reconstruction of SARS-CoV-2 infection in ferrets emphasizes focal infection pattern in the upper respiratory tract

“…Except for genetically modified mice expressing human angiotensin-converting enzyme 2 (ACE2) [39], the presence of SARS-CoV-2 antigen in animal or human brain samples has yet to be shown. 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 [49,52]. Additional immunostaining against tissue-specific cell markers may further facilitate the investigation of the global SARS-CoV-2 cell tropism in affected tissues.…”

“…in light sheet fluorescence microscopy (LSFM) (reviewed in [48]). Lately, the opportunities and advantages of TOC for virus research have been demonstrated in several studies [49–54]. While two approaches to 3D imaging of SARS-CoV-2-infected lung tissue have been described recently [55,56], neither of them is capable of direct visualization of SARS-CoV-2 infection via virus-specific antigen staining.…”

Neuroglia infection by rabies virus after anterograde virus spread in peripheral neurons

“…The advent of efficient full-genome cloning techniques opened novel avenues for the analysis of virus variability in combination with phenotypical characterization of recombinant field viruses at a clonal level [ 44 ]. For example, a recent comparison of field and lab RABV clones in mice revealed that field viruses not only showed a higher virulence compared to lab strains CVS-11, ERA and SAD L16 but also substantially differ from these viruses in their ability to establish infection in non-neuronal astroglia in the central nervous system (CNS) [ 49 ]. In contrast to attenuated lab-adapted RABV, which is supposed to be eliminated from infected glial cells (astrocytes) by type I interferon-induced immune reactions [ 47 ], it has been hypothesized that rapid onset of field virus gene expression in non-neuronal astrocytes blocks inhibitory innate immune activation and responses [ 49 ].…”

“…So far, technical limitations in conventional histology to visualize infection processes in complex tissues in large three-dimensional (3D) volumes at a mesoscopic scale and high resolution hindered a comprehensive overview of RABV cell tropism, in vivo spread, and pathogenesis. Immunofluorescence-compatible tissue clearing iDISCO and uDISCO (immunolabeling-enabled and ultimate three-dimensional imaging of solvent-cleared organs, respectively) protocols [ 46 , 50 ] recently allowed us to perform high-resolutions 3D immunofluorescence analyses of thick RABV-infected tissues slices [ 49 , 70 ].…”