Dapi nuclear counterstain

Virus infectivity was tested by virus isolation from nasal swabs from E1 and E3 between D3 and D28 (D3, D6, D7, D8, D10, D13, D18, D23 and D28). The swab supernatants were diluted 1:25 in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Paisley, Scotland), filtered through a 0.8 μm filter (Sartorius Stedim Biotech, Goettingen, Germany) and added to a monolayer of 4-days-old human rectal tumor cells (HRT-18G, ATTCC CRL-11663) in a 24-well plate. In addition, infective virus was titrated from one nasal swab supernatant using two-fold endpoint dilutions in a 96-well plate. After 1 h incubation at 37 °C, the inoculum was replaced with DMEM with 1 % fetal calf serum and antibiotics (5000 IU penicillin and 5 mg streptocillin/ml). After two days at 37 °C and 5 % CO₂, the cells were fixed with Intracellular Fixation buffer (eBiosience, CA, USA) and stained with 1:80 dilution of monoclonal mouse anti-coronavirus antibody labelled with fluorescein isothiocynate (BioX Diagnostics, Rochefort, Belgium) and DAPI nuclear counterstain (Thermo Fischer Scientific). The wells were observed under a fluorescent microscope for antigen positive cells.

Coronal sections (30 μm thickness) were blocked with 5% (w/v) skim milk powder, 1% bovine serum albumin, and 0.3% Triton X-100 (v/v) in 1× PBS for 1 hour at room temperature in a humidified chamber. Subsequently, the blocking solution was aspirated off the slides, and primary antibodies, GFAP (C9205, Cy3-conjugated, 1:1000), Iba-1 (PA5-21274; 1:500), and CD68 (4-0681-80; 1:300; all from Thermo Fisher Scientific), diluted with fresh blocking solution, were applied onto the tissue sections. Primary antibodies were incubated in a light-shielded humidified chamber and placed in a 4°C fridge overnight. Slides were washed with 1× PBS 3 times for 10 minutes each. Secondary antibodies (A-21244 goat anti-rabbit IgG Alexa Fluor 647 and A-11006 goat anti-rat IgG Alexa Fluor 488; 1:1000; both from Invitrogen, Thermo Fisher Scientific) and DAPI nuclear counterstain (1:500, Invitrogen, Thermo Fisher Scientific) were diluted in fresh blocking solution and applied to the tissue sections to be incubated at room temperature for 1–2 hours. Again, slides were washed with 1× PBS 3 times for 10 minutes each and subsequently coverslipped using Mowiol aqueous mounting medium (MilliporeSigma).

Spinal cord segments from the 28 day survival cohort were cut and stored as described above. Briefly, the slides were rehydrated for 40 min in 0.1 M Tris (pH 7.4). Slides were then washed for 10 min in Tris/0.1% Triton for tissue permeabilization, washed twice for 5 min in Tris, and then 250 μl of fluorescent Nissl neurotracer (1:20, Invitrogen) was added to the sections and incubated in the dark for 20 min. Unbound stain was removed with Tris/0.1% Triton. Slides were washed three times and cover-slipped with Prolong gold antifade reagent with DAPI nuclear counterstain (Invitrogen).

Gut tissue samples were fixed in 4% paraformaldehyde and paraffin-embedded. Tissue sections (5 μm) were treated with antigen retrieval buffer at 100°C (DAKO Target Antigen Retrieval Solution pH 6.1, S2375), blocked for 1 hour at room temperature, and incubated with the following primary antibodies: anti-CD20 (monoclonal mouse anti-human; 1:100; DAKOCytomation, M0755), anti-CD3 (polyclonal rabbit anti-human; 1:100; DAKOCytomation, A0452), and anti–PD-1 (goat anti-human; 1:50; R&D Systems, Alexa Fluor 1086). After overnight incubation at 4°C, slides were washed and stained with secondary antibodies: Alexa Fluor 488 donkey anti-rabbit (Invitrogen, A21206), Alexa Fluor 555 donkey anti-goat (Invitrogen, A21432), and Alexa Fluor 647 donkey anti-mouse (Invitrogen, A31571) for 1 hour at room temperature. Cell nuclei were visualized using DAPI nuclear counterstain (Invitrogen, D1306) and by mounting slides using ProLong Gold Antifade Mountant (Thermo Fisher Scientific).