Vero e6 cells

Parts of infected livers were weighed, homogenized using ceramic beads, and centrifuged for 10 min at 450 g at 4°C. Supernatant was serially diluted in Dulbecco’s modified Eagles Medium (DMEM, Lonza) supplemented with 10% FCS before inoculation onto overnight grown 90% confluent VeroE6 cells in 6 well plates (Corning). After 1 hour incubation, each 10^2 to 10^7 serial diluted inocula was removed and the cell layers were covered with a solution of 0.5% SeaKem®Agarose (Lonza) in Minimal essential Medium Eagle (EMEM, Lonza) supplemented with 10% FCS. The agarose layer was removed after 5 days incubation at 37°C, 5% CO₂, and cells were stained with a crystal violet solution in 2% formaldehyde (Merck). The number of plaques at each dilution was counted and averaged to obtain the viral concentration of the sample in PFU/ml. Each sample was corrected for liver weight input and is expressed as PFU per gram liver.

Vero E6 cells were purchased from the American Type Culture Collection. Vero E6 cells were grown in complete minimal essential media (c-MEM) (Corning, NY, USA) which included 10% FBS (Gibco, Waltham, MA, USA), 5 mM penicillin/streptomycin (Gibco), and l-glutamine (Gibco). Cells were incubated at 37°C with 5% CO₂. DOBV and PUUV were provided by Dr. Connie Schmaljohn (USAMRIID, Frederick, MD, USA). Cells were infected with 0.1 MOI of PUUV or DOBV and grown for 7 days. Virus-infected cells were harvested, aliquoted and grown overnight on 10 spot microscope slides (Fisher) and fixed in acetone as previously described (Jonsson et al., 2010a ). To identify sera with antibody to hantaviral antigens, we first screened a 1:10 dilution of sera in PBS. The presence of antigen-linked antibody was detected by a second incubation with Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher). Samples were read for the presence of perinuclear specific staining using a fluorescent microscope. For sera that were positive for antibodies, we further assessed its reciprocal titer with two-fold dilutions of sera from 1:32 to 1:2048. Slides without virus were prepared and used as a negative control in the evaluation of the IFA slides along with a positive control sera from rodents. In addition, on each slide, well for a negative (no sera) control.

Vero E6 cells (USAMRIID) were plated at 20,000 cells per well in black-walled 96-well plates (Corning 3904). mAbs were serially diluted 3-fold with a maximum of eight dilution spots. Diluted antibodies were mixed with 85 PFU/well of recombinant SARS-CoV-2-nLuc virus, and the mixtures were incubated at 37 °C with 5% CO₂ for 1 hour. Following incubation, growth media was removed, and virus-antibody mixtures were added to the cells in duplicate. Virus-only controls were included in each plate. Following infection, plates were incubated at 37 °C with 5% CO₂ for 48 hours. After the 48-hour incubation, cells were lysed, and luciferase activity was measured via Nano-Glo Luciferase Assay System (Promega) according to the manufacturer specifications. Neutralization titers were defined as the sample dilution at which a 50% reduction in relatively light unit (RLU) was observed relative to the average of the virus control wells.

Vero E6 cells (ATCC CRL-1586) were plated in a T225 flask with complete DMEM (Corning 15-013-CV) containing 10% FBS, 1×PenStrep (Corning 20-002-CL), 2 mM l-Glutamine (Corning 25-005-CL) overnight at 37 °C 5% CO₂. The media in the flask was removed and 2 mL of SARS-CoV-2 strain USA-WA1/2020 (BEI Resources NR-52281) in complete DMEM was added to the flask at an MOI of 0.5 and allowed to incubate for 30 min at 34 °C 5% CO₂. After incubation, 30 mL of complete DMEM was added to the flask. The flask was then placed in a 34 °C incubator at 5% CO₂ for 5 days. On day 5 post-infection the supernatant was harvested and centrifuged at 1000×g for 5 min. The supernatant was filtered through a 0.22 µm filter and stored at −80 °C.