Cercopithecus aethiops

Vero E6 cells (C1008; African green monkey kidney cells) were obtained from Public Health England and maintained in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS) and 0.05 mg/mL gentamycin at 37°C with 5% CO₂. SARS-CoV-2 isolate SARS-CoV-2/human/Liverpool/REMRQ0001/2020, which was cultured from a nasopharyngeal swab from a patient, was passaged a further 4 times in Vero E6 cells. The fourth passage of virus was cultured in Vero E6 cells with DMEM containing 4% FBS and 0.05 mg/mL gentamycin at 37°C with 5% CO₂ and was harvested 48 hours post inoculation. Virus stocks were stored at −80°C.

Vero E6 (African green monkey kidney, kind gift from Peter Bredenbeek, LUMC, NL) and HuH7 (human hepatoma, JCRB0403) cells were maintained in minimal essential medium (Gibco) supplemented with 10% fetal bovine serum (Integro), 1% bicarbonate (Gibco), and 1% L-glutamine (Gibco). For maintenance of Calu-3 cells (human airway epithelium, kind gift from Lieve Naesens, KU Leuven, BE), the above medium was supplemented with 10 mM HEPES (Gibco). All assays involving virus growth were performed using 2% (Vero E6 and HuH7) or 0.2% (Calu-3) fetal bovine serum instead of 10%.
SARS-CoV-2 strain BetaCov/Belgium/GHB-03021/2020 (EPI ISL 407976|2020-02-03) recovered from a nasopharyngeal swab taken from a reverse transcription quantitative polymerase chain reaction (RT-qPCR)-confirmed asymptomatic patient returning from Wuhan, China beginning of February 2020⁷⁵ was directly sequenced on a MinION platform (Oxford Nanopore)^{76 (link)}. Phylogenetic analysis confirmed a close relation with the prototypic Wuhan-Hu-1 2019-nCoV (GenBank accession number MN908947.3) strain. Infectious virus was isolated by serial passaging on HuH7 and Vero E6 cells (see Supplementary Fig. 1), with the addition of penicillin/streptomycin, gentamicin, and amphotericin B. Virus used for animal experiments was from passages P4 and P6. Prior to inoculation of animals, virus stocks were confirmed to be free of mycoplasma (PlasmoTest, InvivoGen) and other adventitious agents by deep sequencing on a MiSeq platform (Illumina) following an established metagenomics pipeline^{77 (link),78} . The infectious content of virus stocks was determined by titration on Vero E6 cells by the Spearman–Kärber method. All virus-related work was conducted in the high-containment BSL3+ facilities of the KU Leuven Rega Institute (3CAPS) under licenses AMV 30112018 SBB 219 2018 0892 and AMV 23102017 SBB 219 2017 0589 according to institutional guidelines.
Antibody VHH-72-Fc was administered i.p. at a dose of 20 mg/kg 1 day prior to infection. VHH-72-Fc was expressed in ExpiCHO cells (ThermoFisher Scientific) and purified from the culture medium as described²³ . Briefly, after transfection with pcDNA3.3-VHH-72-Fc plasmid DNA, followed by incubation at 32 °C and 5% CO₂ for 6–7 days, the VHH-72-Fc protein in the cleared cell culture medium was captured on a 5 ml MabSelect SuRe column (GE Healthcare), eluted with a McIlvaine buffer pH 3, neutralized using a saturated Na₃PO₄ buffer, and buffer exchanged to storage buffer (25 mM L-Histidine, 125 mM NaCl). The antibody’s identity was verified by protein- and peptide-level mass spectrometry.

MRC-5 cells (human lung fibroblasts, ATCC-CCL-171) were cultured essentially as described previously [30 (link)]. Vero E6 cells (African green monkey kidney epithelial cells), 293/ACE2 cells (originally described to be derived from human HEK293 cells [95 (link)], but most likely from nonhuman primate origin [14 (link)]) and BHK-21 cells (baby hamster kidney fibroblasts) were cultured essentially as described previously [21 (link)]. Vero cells (ATCC-CCL81) cells were cultured essentially as described previously [96 (link)]. All culture media contained 100 IU/ml penicillin and 100 ug/ml streptomycin unless otherwise specified.
CHIKV LS3 is a synthetic Chikungunya virus based on the consensus sequence of E1-226V isolates [21 (link)]. Infections with CHIKV LS3 and LS3-GFP were performed essentially as described previously [21 (link)]. The Chikungunya replicon was derived from CHIKV LS3 by replacing the structural genes with a puromycin resistance/foot-and-mouth disease virus 2A oligopeptide/green fluorescent protein (PAC-2A-GFP) reporter gene. The sequence of the 2A oligopeptide contains an amino acid motif that prevents formation of the peptide bond between glycine and the final proline of the sequence which allows expression of multiple proteins from a single ORF [97 (link),98 (link)].
Vero E6 cells were infected with the Sindbis virus (SINV) HR-strain [99 (link)], Semliki Forest virus (SFV) strain SFV4 [100 (link)] or VEEV vaccine strain TC-83 [101 (link)] at a multiplicity of infection (MOI) of 5 or 10. Vero E6 cells were infected with a GFP-expressing recombinant human adenovirus type 5 (HAdV-GFP/LUC; [102 (link)]) at an MOI of 5 and with a GFP-expressing recombinant coxsackie B3 virus (CVB3) (a kind gift from prof. dr. Frank van Kuppeveld, Utrecht University) at an MOI of 1. BHK-21 cells were infected with the equine arteritis virus (EAV) Bucyrus strain at MOI 5 at 39.5°C essentially as described previously [103 (link)].