**1. Introduction**

The rabies virus (RABV) is a highly neurotropic virus, which inevitably causes lethal disease in mammals after onset of neurological signs [1]. As a non-segmented, single-stranded RNA virus of negative RNA polarity, RABV belongs to the *Rhabdoviridae* family in the order *Mononegavirales* [2]. With nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, and the large polymerase L, the 12 kb genome of RABV encodes five virus proteins, all of which are essential for virus replication and spread [3]. In addition to essential roles of the virus proteins in genome replication and virus assembly, multiple accessory functions of the RABV proteins have been identified. RABV pathogenicity has mainly been attributed to a potent interference with the innate immune system by N, P, and M [4–10], and neuronal survival regulation by G [11–14]. 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-synaptic tracing [22,23] demonstrate that the general capacity of axonal transport and trans-synaptic spread cannot explain mechanistic differences between highly virulent RABV and more attenuated lab strains. Differences between RABV lab strains in the efficiency of trans-synaptic spread [24] indicate that the efficacy of the involved processes, more than the capability itself, may contribute to RABV spread in vivo.

With nAChR (nicotinic acetylcholine receptor), NCAM (neuronal cell adhesion molecule), p75NTR, and mGluR2 (metabotropic glutamate receptor subtype 2) supporting RABV entry [25–28], several RABV receptors have been discussed. However, none of these receptors are essential for CNS infection by RABV, and a broad panel of non-neuronal cell types can be infected in vitro [29], indicating that cell tropism of RABV is not restricted to neurons by receptor specificity.

Most in vivo studies report a strict neurotropic infection. Directly after exposure by bite, however, muscle cells are infected (reviewed in [30]), and infection of non-neuronal cells in the CNS can occur [29,31,32]. Use of recombinant Cre recombinase-expressing RABV led to the identification of abortively infected glial cells in infected mouse brains, strongly suggesting infection of and virus elimination from these cells by a potent type I interferon response [33]. Accordingly, abortive infection of non-neuronal cells and induction of innate immune responses may play an important role in the infection process itself and in regulating downstream adaptive immune pathways. Indeed, a model based on in vitro-infected astrocytes suggests that, in contrast to wild-type RABV, attenuated RABV activates inflammatory responses in astrocytes through increased double-strand RNA (dsRNA) synthesis and recognition by retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated protein 5 (MDA5) [34]. Highly virulent field RABV isolates are able to evade or at least delay host immune reactions [35], which may allow virus replication to reach pathogenic levels, whereas early innate immune induction via astrocytes or other glial cells by attenuated viruses does not. Nevertheless, all productive RABV infections eventually cause rabies as a disease, which is always associated with an encephalitis. However, differences appear to exist as field viruses cause less tissue damage and neuroinflammation than attenuated lab RABV [34,36,37].

To compare the cell tropism of highly virulent field and lab-adapted RABVs in the CNS, we employed the novel immunostaining-compatible tissue clearing technique uDISCO (ultimate 3D imaging of solvent-cleared organs) [38–40] for detection and quantification of RABV infection in neurons and astrocytes in solvent-cleared brain tissue. After confocal laser scan acquisition of large confocal z-stacks in thick tissue slices, three-dimensional (3D) reconstructions were performed to visualize the cellular context of RABV infection. Frequencies of RABV infection in neurons and astrocytes were determined after intramuscular (i.m.) and intracerebral (i.c.) inoculation, leading to novel insights regarding the ability to establish RABV infection in non-neuronal astrocytes, its correlation with RABV virulence, and its dependence on the infection route. These data strongly support a model in which, contrary to in vitro conditions where all viruses are able to infect non-neuronal astrocytes, there are differences between field and lab-adapted viruses in their ability to replicate to detectable levels in astrocytes in vivo. The infection of astrocytes with virulent field RABV and the accumulation of interferon antagonistic virus proteins may block rapid innate immune induction, thereby contributing to immune evasion, even in the context of glial cell infection.

### **2. Materials and Methods**

### *2.1. Cell Lines, Primary Brain Cell Preparation, and Cultivation*

Mouse neuroblastoma cells (Na42/13) were used for virus amplification. All cells were provided by the Collection of Cell Lines in Veterinary Medicine (CCLV), Friedrich-Loeffler-Institut, Insel Riems, Germany. Primary neurons and astrocytes were prepared from 1-day-old neonatal Sprague Dawley rats (P0–P1) [41]. The rats were decapitated, the heads were disinfected in 70% ethanol, the brains were removed, and the hippocampi were separated and mechanically minced. The hippocampal cells were transferred into ice-cold Hank´s Balanced Salt Solution (HBSS) and stored on ice. After preparation, the brain cells were dissociated by adding 0.25% trypsin + EDTA (Gibco; Thermo Fisher Scientific, Darmstadt, Germany) and DNase I (Applichem, Darmstadt, Germany) and incubated for 15 min at 37 ◦C. Afterwards, further dissociation in Neurobasal-A Medium (NB-A, Gibco; Thermo Fisher Scientific) was performed by using a glass Pasteur pipette with a fire-polished tip. The cells were purified with an OptiprepTM gradient (with concentrations from bottom to the top: 17.3%, 12.4%, 9.9%, and 7.4%) and centrifuged for 15 min at 800× *g* without brake. Next, the cells were washed with NB-A medium and counted with the LUNA-II Automated Cell Counter (Logos Biosystems, Villeneuve d'Ascq, France). The neuronal cells were seeded on coverslips in a 24-well plate and cultured for two weeks at 37 ◦C and 5% CO2 in serum-free NB-A supplemented with 2% B27 (Gibco; Thermo Fisher Scientific), 1% GlutaMAX (Gibco; Thermo Fisher Scientific), 1% Penicillin-Streptomycin (stock: 10,000 U/mL penicillin and 10 mg/mL streptomycin; Sigma-Aldrich, Taufkirchen, Germany), and 0.2% Gentamicin (stock: 50 mg/mL; Gibco; Thermo Fisher Scientific) until they were used for infection experiments.
