Dapi staining

Cell staining was done on GSCs seeded on multichamber slides (Nunclone, Sigma-Aldrich). Cells were washed in PIPES buffer (80 mM PIPES pH 6.8, 5 mM EGTA and 2 mMMgCl2, Sigma-Aldrich, MO, USA), and fixed with 4% paraformaldehyde/PIPES (Sigma-Aldrich) for 10 min. This step was followed by block-permeabilization in PBS containing 0.2% BSA (Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich) for 10 min, followed by DAPI staining (Sigma-Aldrich). The antibodies were used at the condition suggested by the suppliers: mouse anti-beta3 tubulin (T8578, 1/100,Sigma)-, rabbit Glial Fibrillary Acidic Protein (GFAP, Z0334, 1/100, Dako, CA, USA), and mouse anti-Flavivirus (Ab10216,1/1000, Abcam, Cambridge, UK).
All microscopic images were acquired with a Nikon system TE2000-S microscope (Nikon Instruments, Amsterdam, Netherlands) equipped with an Olympus LC20 Camera and Olympus soft imaging LCmicro software (Olympus, Milan, Italy), or with a Leica confocal station (Leica SP5 confocal system, mounted on a Leica DM6000 inverted microscope, equipped with an Argon-ion laser and PMT detectors) (Leica Microsystems, Wetzlar, Germany).

Vero cells were seeded on cover slips, infected with viruses in BSL4 condition and fixed using 4% paraformaldehyde (PFA) for 48 h, and then for additional 24 h with fresh 4% PFA, before removal from the BSL4 laboratory and transfer to the BSL2 laboratory for immunostaining, mounting on slides and imaging on an Olympus IS71 microscope, as previously described71 (link). Viral antigens were detected using either anti-RVFV NS mouse polyclonal antibody, anti-RSSEV Sophy mouse polyclonal antibody or anti-CCHFV rabbit polyclonal antibody (kindly provided by Drs. Tesh and Ksiazek, World Reference Center for Emerging Viruses and Arboviruses, UTMB), coupled to a secondary goat anti-mouse or anti-rabbit antibody (Thermo Fisher Scientific). Cells infected with EBOV-eGFP were directly imaged in the BSL4 laboratory. DAPI staining (Sigma-Aldrich) was added to localize cell nuclei in fixed samples.

To test p65 nucleus translocation by immunofluorescence (IF), RAW264.7 macrophages were seeded on confocal dishes overnight and incubated with LPS in the presence or absence of GRb3 for 1 h. After washing with PBS for 10 min, paraformaldehyde was employed to immobilize cells, followed by blocking cells with goat serum containing 0.1% Triton. Then, macrophages reacted with p65 antibody overnight, which was diluted at 1:400 with 5% goat serum solute. After incubation with Alexa Flour 568-combined IgG (Thermo Fisher, #A-21069) for 2 h, cells were subjected to DAPI staining (Beyotime) for 5 min. In addition, to detect whether GRb3 blunted LPS binding to the cell membranes, Alexa Fluor 488-conjugated LPS (LPS488, Sigma Aldrich, 10 μg/ml) was exposed to macrophages for 12 h. PBS was adopted to rinse cells three times, with DAPI staining for 10 min. Subsequently, a confocal microscope (CarlZeiss, Germany) was employed to determine p65 nucleus transfer and LPS uptake to cytomembranes.

IgG subclasses of mimotope-immunized rat sera were investigated using a live cell-based-assay with HEK293 cells expressing rat-AQP4 fused to EmGFP [31 (link)]. Sera from four different animals were studied. The mouse monoclonal AQP4 antibody E5415A [32 (link)] was used as a positive control. Sera taken from a mimotope-immunized rat with an antibody titer of zero and from a normal healthy control rat served as negative controls.
HEK293 cells expressing rat AQP4-EmGFP were blocked with goat-IgG (4 μg/ml; Sigma-Aldrich) and subsequently incubated with rat sera diluted 1:20 and 1:40 in 10%FCS/PBS (FCS, Gibco; PBS, Sigma-Aldrich) or with the mouse monoclonal AQP4 antibody E5415A (5 μg/ml in 10%FCS/PBS) for 1 h at 4 °C. After washing three times with 10%FCS/PBS, IgG subclasses were determined by incubating with mouse anti-rat IgG1, IgG2a, IgG2b and IgG2c antibodies (1:200 in 10%FCS/PBS; BioLegend) for 30 min at 4 °C. Following a washing step cells were incubated with Alexa Fluor 594 labelled goat anti-mouse IgG (1:500 in 10%FCS/PBS; Invitrogen) at room temperature for 30 min. Dead cells were excluded by DAPI staining (Sigma-Aldrich). Data were analyzed by two investigators (ML and KS).

Log phase cells (5 × 10³ cells per well) were seeded in 96-well plates. The cell proliferation was assessed with Cell Counting Kit-8 (CCK8) according to the manufacturer’s instructions. Proliferation rates were determined at 0, 24, 48, 72 and 96 h after transfection by measuring the absorbance. The Cells in each group were tested for 5 replicates. The EDU Cell Proliferation Assay Kit (Green Sky, Shanghai, China) was used to evaluate cell proliferation. At the log phase of cell growth, the cells were spiked with 5-ethynyl-29-deoxyuridine (EdU) according to the manufacturer’s instructions. Briefly, the cells were incubated with 50 mM EdU for 3 h, and then the cellular nuclei were treated with DAPI staining (Sigma) at a concentration of 1 mg/ml for 10 min, the cell proliferation indexes were photographed and recorded by fluorescence microscopy.

Monkeys were euthanized (1 ml ketamine, 0.15 g/ml, i.m.) for perfusion with 500 ml saline and 500 ml 10% formaldehyde in PBS. The brains were removed and fixed again in formalin for 3–4 days, and then equilibrated with 20% and 30% sucrose. Coronal sections of the substantia nigra were obtained by sectioning the tissue on a freezing microtome (Leica, CM1850UV-1-1). Slice thickness was set at 18 μm. Slices were first blocked with 10% sheep serum for 45 min at room temperature and then incubated with primary antibody overnight at 4 °C. Primary antibodies are listed in the following: NeuN (Neuronal Nuclei) (Millipore, MAB377, 1:100); Iba1 (Wako Pure Chemical Industries Ltd, 019–19741, 1:300); Tyrosine Hydroxylase (TH) (Millipore, AB152, 1:400); Doublecortin (DCX) (Millipore, MABN707, 1:200); GFAP (Sigma, G9269, 1:2000). The following day, cells were washed three times with PBS media and incubated with Alexa 488 or Rhodamine-conjugated secondary antibodies (Invitrogen: goat-anti-rabbit, goat-anti-mouse, donkey- anti-goat, donkey-anti-chicken, 600×) in PBS for one hour at RT. Nuclei were visualized with DAPI staining (Sigma-Aldrich).