Fig 2: Spinal column infection after i.m. and i.c. infection. a, b Maximum z-projections of light sheet overviews of cross sections of the spinal column after i.m. (a) and i.c. (b) infection [2.0x magnification; z = 824 µm (a), 1480 µm (b)]. Red: RABV P; blue: nuclei. Green fluorescence for NEFM not shown as separation from green autofluorescence was not possible at low resolution. Stars: grey matter, triangles: white matter. Scale bar: 1500 µm. c Maximum z-projection of detail from (a), including NEFM signals (green). Scale bar: 200 µm. d, e Maximum z-projection of confocal z-stacks of infected spinal cord (d) [z = 31 µm; Scale bar: 100 µm] and detail (e) [z = 10 µm; scale bar: 10 µm]. f Maximum z-projection of detail from (a) with neurons projecting from the spinal cord. Scale bar: 200 µm. g, h Maximum z-projection of confocal z-stacks (g) [z = 36 µm, Scale bar: 100 µm] and detail (h) [z = 10 µm; Scale bar: 10 µm] of projecting nerves from the spinal cord. i Maximum z-projection of detail from (b), including NEFM signals (green). Scale bar: 200 µm. j, k Maximum z-projection of confocal z-stacks of infected spinal cord (j) [z = 61 µm; Scale bar: 100 µm] and detail (k) [z = 20 µm; scale bar: 10 µm]. l Maximum z-projection of detail from (B), including NEFM signals (green). Scale bar: 200 µm. m, n Maximum z-projection of confocal z-stacks of projecting nerve (m) [z = 41 µm; Scale bar: 100 µm] and detail (n) [z = 20 µm; scale bar: 10 µm]

Fig 3: RABV P in axons and infected Schwann cells in coronal head sections. a Maximum z-projection of high-resolution confocal z-stacks of mouse head sections after i.c. inoculation and staining for RABV P (red), NEFM (green) and nuclei (blue) [z = 38 µm]. Arrows indicate RABV P surrounding NEFM. Scale bar: 100 µm. b–e Maximum z-projections (b, d) and corresponding 3D projections (c, e) of details of (a) (white boxes). f Maximum z-projection of confocal z-stacks of mouse head sections after i.c. inoculation stained for RABV P (red), MBP (green) and nuclei (blue) [z = 29 µm]. Scale bar: 25 µm. g–i Single slices of (f). Arrowhead: RABV P surrounding MBP-positive myelin sheath. Star: RABV P surrounded by MBP-positive myelin sheath. Scale bar: 5 µm. j 3D projection of (g–h)

Fig 4: RABV distribution in head cross sections after i.m. inoculation. a, b Maximum z- (a) and 3D projection (b) of coronal mouse head sections after i.m. infection with field RABV rRABV Dog [1.26x magnification; z = 4210 µm]. Indirect immunofluorescence staining against RABV P (red), NEFM (green), and nuclei (blue) revealed a markedly reduced degree of infection after i.m. infection in comparison to i.c. infection (see Fig. 5). c, e, g, i Maximum z-projections of details from A (white boxes) [12.6x; magnification] (Scale bar: 200 µm). d, f, h, j Confocal high-resolution z-stacks (corresponding to c, e, g, i, respectively) [z = 80 µm (d), 26 µm (e), 72 µm (h), 74 µm (j); scale bar: 100 µm] with details (scale bar: 15 µm). c, d infected eye, e, f nasal epithelium, g, h tongue, i, j and teeth in mouse head coronal section

Fig 5: Anterograde virus spread: RABV in Schwann cells of a hind leg nerve after i.c. inoculation. a, b Maximum z-projection of light sheet overview (a) and detail (b) of hind leg section after staining for RABV P (red), and nuclei (blue). Green fluorescence for NEFM is not shown as separation from green autofluorescence was not possible at low resolution. a Magnification of 1.6x [z = 1944 µm; Scale bar: 1500 µm] and b magnification of 12.6x [z = 58 µm; Scale bar: 200 µm]. c Maximum z-projection of confocal z-stack [z = 37 µm] of infected nerve fiber from (b), now including NEFM staining (green). Scale bar: 100 µm. d, e Maximum z-projection of details from (c) (see white boxes) [z = 5 µm (d, e)]. Scale bar: 10 µm. f, g Maximum z-projection of hind leg nerve section stained for RABV P (red), MBP (green) and NEFM (blue) [z = 15 µm]. To visualize low amounts of viral antigen, the MBP channel is excluded in (g). Scale bar: 50 µm. h 3D projection of detail from (f) (see white box). i–k Single planes from detail of (f). The arrowhead indicates an RABV-infected cell. Scale bar: 5 µm