*2.3. Antibodies*

To verify the presence of RABV, a polyclonal rabbit serum against recombinant RABV P protein (P160-5, immunofluorescence 1:5000, uDISCO 1:3000) and a polyclonal goa<sup>t</sup> serum against RABV N (goat anti-RV N, immunofluorescence 1:4000), which have been described previously [40,50], were used. The polyclonal chicken anti-glial fibrillary acidic protein (GFAP) antibody (Thermo Fisher Scientific, Darmstadt, Germany; #PA1-10004, uDISCO 1:1500), the polyclonal rabbit anti-GFAP (Dako, #Z0334, immunofluorescence 1:500), the polyclonal guinea pig anti-NeuN antibody (Synaptic Systems, Goettingen, Germany; #266004, uDISCO 1:800), and the rabbit anti-MAP2 antibody (Abcam, Cambridge, UK; #ab32454, immunofluorescence 1:250) were purchased from their respective suppliers.

### *2.4. Infection of in Vitro Cell Cultures and Immunofluorescence Staining*

Two-week-old primary rat hippocampal cell cultures were infected with 1 × 10<sup>3</sup> infectious units of rRABV Dog, rRABV Fox, rCVS-11, and SAD L16, and cultivated for 24 h at 37 ◦C and 5% CO2. Indirect immunofluorescence was performed by standard techniques after fixation with 4% paraformaldehyde (PFA) in phosphate-bu ffered saline (PBS) for 30 min and 15 min permeabilization with 0.5% Triton X-100 in PBS. Afterwards, samples were blocked with 0.025% skim milk powder in PBS for 15 min. Immunostainings were executed by 1.5 h incubation with primary antibodies, three wash steps with PBS, followed by 1 h incubation with secondary antibodies and additional Hoechst33342 (1 μg/mL) for staining of the nuclear chromatin. Specimens were mounted on coverslips and were analyzed by confocal laser scanning microscopy.

### *2.5. Brain Samples and Mouse Infections*

Three- to four-week-old BALB/c mice (Charles-River, Germany) were infected with rRABV Rac, rCVS-11, ERA, and SAD L16 viruses using two di fferent inoculation routes and two di fferent viral doses, essentially as described before [51]. Two groups of six animals each were anesthetized and infected i.m. with 10<sup>2</sup> or 10<sup>5</sup> TCID50/30 μL, and an additional group of three mice was infected i.c. with 10<sup>2</sup> TCID50/30 μL. The weight and the clinical score, ranging from zero to four, of all mice were observed for 21 days post infection (dpi). When reaching a clinical score of two or three (ru ffled fur, slowed movement, weight loss >15%), the animals were anaesthetized with isoflurane and euthanized through cervical dislocation. Samples were taken, fixed with 4% paraformaldehyde (PFA) for one week, and stored for further processing. All remaining animals were euthanized at 21 dpi. Mouse experimental studies on the characterization of lyssaviruses were evaluated by the responsible animal care, use, and

ethics committee of the State Office for Agriculture, Food Safety, and Fishery in Mecklenburg-Western Pomerania (LALFF M-V) and gained approval with permissions 7221.3–2–001/18.

### *2.6. Archived Mouse Brains Infected with rRABV Dog and rRABV Fox*

To minimize animal experiments, archived PFA-fixed brains from previous pathogenicity trials [42] were used for the analysis of rRABV Dog and rRABV Fox virus infections in mice. Similar to the mouse experiments with rRABV Rac, rCVS-11, ERA, and SAD L16 described above, mice were inoculated via the i.c. or the i.m. route.

### *2.7. Ultimate 3D Imaging of Solvent-Cleared Organs (uDISCO)*

The clearing of brain tissue slices was performed as described previously [40] in modification of earlier publications [38,39]. Briefly, the PFA-fixed tissues were sectioned into 1 mm-thick slices using a vibratome (Leica VT1200S; Leica Biosystems, Wetzlar, Germany). All subsequent incubations steps were performed with gentle oscillation. To increase antibody diffusion and reduce tissue autofluorescence [38], the sections were pretreated with increasing concentrations of methanol (20%, 40%, 60%, 80%, and twice in 100%; dilutions with distilled water, incubation for 1 h each) and bleached by overnight incubation at 4 ◦C with 5% H2O2 in 100% methanol. After removal of the bleaching solution, the samples were rehydrated with decreasing concentrations of methanol (80%, 60%, 40%, and 20%; dilutions with distilled water, incubation for 1 h each) and a subsequent wash with PBS for 1 h. To permeabilize the samples, they were washed twice for 1 h each with 0.2% Triton X-100 in PBS and subsequently incubated for 48 h at 37 ◦C in 0.2% Triton X-100/20% DMSO/0.3 M glycine in PBS. The samples were then blocked by incubation with 0.2% Triton X-100/10% DMSO/6% donkey serum in PBS for 48 h at 37 ◦C. Primary antibodies were diluted in 3% donkey serum/5% DMSO in PTwH (0.2% Tween-20 in PBS with 10 μg/mL heparin), and incubation of samples was performed at 37 ◦C for 5 days. The antibody solution was refreshed after 2.5 days. The samples were washed with PTwH by exchanging the solution four times during the course of the day and subsequently incubated overnight in PTwH. Incubation with secondary antibodies was performed in 3% donkey serum in PTwH for 5 days at 37 ◦C, refreshing the secondary antibody solution once after 2.5 days. Subsequent washing was performed as described above following primary antibody incubation.

For tissue clearing, the samples were dehydrated with a series of *tert*-butanol (TBA) solutions (30%, 50%, 70%, 80%, 90%, and 96%; dilutions with distilled water, incubation for 2 h each), leaving 96% TBA on overnight. Following further dehydration in 100% TBA for 2 h, the samples were cleared in BABB-D15 [39] [1:2 mixture of benzyl alcohol (BA) and benzyl benzoate (BB), which is mixed with diphenyl ether (DPE) at a ratio of 15:1 and supplemented with 0.4 vol% DL-α-tocopherol] until they were optically transparent (2–6 h).

For confocal laser scanning microscopy, the samples were mounted in 3D-printed imaging chambers (printer: Ultimaker 2 + [Ultimaker, Utrecht, Netherlands], material: co-polyester, nozzle: 0.25 mm, layer height: 0.06 mm, wall thickness: 0.88 mm, wall count: 4, infill: 100%, no support structure; the corresponding .STL file is provided in the Supplementary Materials of Zaeck et al. [40]).

### *2.8. Confocal Laser Scanning Microscopy and Image Processing*

The immunofluorescent staining of primary brain cells and infected tissues was visualized with a confocal laser scanning microscope (Leica DMI 6000 TCS SP5; Leica Microsystems, Wetzlar, Germany) equipped with a long free working distance 40× water immersion objective (NA = 1.1; Leica, #15506360). For image processing, a Dell Precision 7920 workstation was used (CPU: Intel Xeon Gold 5118, GPU: NvidiaQuadroP5000,RAM:128GB2666MHzDDR4,SSD:2TB;Dell,FrankfurtamMain,Germany).

For quantification of infected cells in thick tissue sections, the image was split into individual channels using Fiji, an ImageJ (v1.52h) distribution package [52]. A bleach correction was performed (simple ratio; background intensity: 5.0), and brightness and contrast were adjusted for each channel. Objects were identified and counted with the 3D Objects Counter plugin [53]. The resulting objects

map was overlaid with the RABV P channel to quantify infected objects. For each sample, at least six regions were imaged and analyzed. The 3D projections in Figure 2d,e were generated with Icy [54]. All other maximum z- and 3D projections, including the Supplementary Videos S1–S4, were generated with Fiji.
