293f suspension cells

Jurkat T cell line (clone E6.1) was obtained from ATCC and 293T cells from Cell Genesys (Foster City, CA). Jurkat cells and primary T cells were maintained in RPMI-1640 and 293T cells were maintained in Dulbecco’s modified Eagle’s medium. Media were supplemented with 10% heat-inactivated FCS, 2 mM Glutamax, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were grown at 37 °C in 5% CO₂.
Cell viability was determined using a trypan blue exclusion method and counted using the Countess II FL automated cell counter (Invitrogen). 293F suspension cells, obtained from Thermo Fisher Scientific, were maintained in shaker flasks at 120 r.p.m., under 8% CO₂ at 37 °C and propagated in their respective FreeStyle media (serum-free/defined).

Recombinant HA (rHA) proteins were generated using pCMV-Sport6 vectors encoding full-length, codon-optimized HA sequences from A/Astrakhan/3212/2020 (EPI ID 1038924), A/pheasant/New York/22-009066-001/2022 (EPI ID 11971502), A/red fox/England/AVP-M1-21-01/2020 (EPI ID 2081527), A/Vietnam/1203/2004 (EPI ID 116507), A/Hubei/1/2010 (Genbank CY098760.1), A/Indonesia/5/2005 (Genbank CY116648.1), or the “headless” HA stalk of A/Vietnam/1203/2004. For each of these proteins, the HA transmembrane domain was replaced with the Foldon T4 trimerization domain of T4 fibritin, an Avitag site-specific biotinylation sequence, and a hexahistidine tag as described previously^{36 (link)}. To produce the recombinant proteins, rHA plasmid and a plasmid encoding neuraminidase (NA) from A/Puerto Rico/8/1934 were co-transfected into 293 F suspension cells (Thermo Fisher) using 293Fectin (Thermo Fisher). Supernatants were collected 4 days later for protein purification by Ni-NTA affinity chromatography (Qiagen).

RoNi/7.1 cells [Rousettus aegyptiacus immortalized cells from kidney tissue; kindly provided by M. A. Müller and C. Drosten, Charité-Universitätsmedizin Berlin, Germany; (13 (link))] were maintained in RoNi cell medium [Dulbecco's Modified Eagles Medium (DMEM) containing 1% MEM non-essential amino acids solution (100x concentrate), 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate] supplemented with 10% fetal bovine serum (FBS). Culture conditions for 293F suspension cells (human embryo kidney cells, ThermoFisher Scientific) have been described previously (19 (link)). Vero E6 cells (BEI Resources, Cat NR-596) were maintained in DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. VSV-eGFP was a gift from Dr. John H. Connor (Boston University School of Medicine), and was propagated as previously described (19 (link)). Virus stocks for MARV isolates Musoke (GenBank: NC_001608; BEI Resources) and Angola (GenBank: KR867677.1; BEI Resources) were propagated in Vero E6 cells as described previously (64 (link)). Identity of virus extracted nucleic acids was confirmed by deep sequencing. Virus titers were determined in the same cells by plaque assay as described elsewhere (65 (link)). All work with infectious MARV was performed in the BSL-4 facility of the Texas Biomedical Research Foundation, San Antonio, TX.