Trueblue peroxidase substrate

FFA procedures were performed as previously described^{52 (link)}. Tissue samples were homogenized and clarified by centrifugation. Tissue supernatants and sera were diluted serially before infection on BHK cells. BHK cells used in this assay do not express Fcγ receptors. Cells were plated (2.0 × 10⁵ cells/well in a 24-well plate) and incubated overnight at 37 °C, 5% CO₂. Confluent monolayers were inoculated with undiluted or 10-fold serially diluted sera or clarified tissue supernatant, and were incubated for 1 hr at 37 °C. After incubation, the inoculum was removed, and each cell monolayer was overlaid with CMC-media and incubated at 37 °C, 5% CO₂ for 1.5–2 days. Cells were then fixed, permeabilized, and incubated with pan Flavivirus anti-envelope (E) Ab clone 4G2 (BioXCell), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) and staining with True-Blue peroxidase substrate (Sera Care). Foci were counted, virus levels in the serum were expressed as Focus Forming Units (FFU) per mL, and for most tissues as FFU/g. As it was not technically feasible to weigh sciatic nerves, viral levels in these tissues were expressed as FFU/tissue.

Virus samples were titered on Vero cells using infectious center assays (ICA) or focus-forming assays (FFAs). Vero cells were seeded at 1.2 × 10⁴ cells/well in 96-well plates, cultured overnight, and infected with 100 µL of serial dilutions of virus samples for 4 h. The virus inocula were aspirated and replaced with 100 µL of DMEM plus 5% FBS and 20 mM NH₄Cl or 1% carboxymethylcellulose in modified Eagle’s Medium supplemented with 2% heat-inactivated FBS and 10 mM Hepes pH 7.4. For ICA, 48 h post-infection, cells were fixed with 4% paraformaldehyde (PFA, Electron Microscopy Science) and stained with RuV pAb and fluorescently labeled secondary antibody, and infection was quantitated by fluorescence microscopy. For FFA, cells were fixed by adding 100 µL of prewarmed 1% PFA in PBS to the overlay and incubating for 1 h. Cells were washed with PBS, permeabilized with 0.1% saponin in PBS containing 0.1% bovine serum albumin (BSA), incubated with RuV pAb followed by incubation with horseradish peroxidase-conjugated rabbit anti-goat IgG (Seracare, Milford, MA). Foci were developed using TrueBlue Peroxidase substrate (Seracare) and quantified using an ImmunoSpot S6 Macroanalyzer with Biospot 7.0.9.10 software (Cellular Technologies, Shaker Heights, OH). The titer for psVSV-RuV was determined by ICA, with initial infection for 2 h and scoring of eGFP-positive cells 24 h post-infection.

Overnight cultured Vero cells (5 × 10⁴ cells/well) in a 24-well plate were infected with 10-fold serially diluted ZIKV or DENV for 1 h at 37°C. After incubation, the inoculum was removed and cells were immediately replenished with plaque medium supplemented with 0.8% carboxylmethylcellulose (CMC). YFV-17D was titrated in Vero, clone E6 using 0.8% CMC. ZIKV and DENV-infected cells were incubated for 5 and 4 days in a CO₂, respectively. ZIKV and YFV-17D infected cells were fixed and stained with 4% paraformaldehyde and 0.25% crystal violet, respectively. DENV-infected cells were fixed and immunostained with mouse anti-DENV envelope antibody followed by HRP-conjugated anti-mouse antibody. The foci were developed using TrueBlue peroxidase substrate (Sera Care, USA).
For human influenza viruses, overnight cultured MDCK cells (2 × 10⁵ cells/well) in a 12-well plate were infected with serially diluted influenza viruses for 1 h at 37°C. Following incubation, the inoculum was removed and replenished with DMEM supplemented with 0.3% BSA, 25 mM HEPES, 1 µg/ml TPCK-treated trypsin and 0.8% Avicel. At 2 days post-infection, infected cells were fixed and stained with 4% paraformaldehyde and 0.25% crystal violet, respectively.

FRNT assay was performed as described recently^{54 (link)}. Briefly, HeLa-ACE2 cells were seeded in 12 μL complete DMEM at a density of 2 × 10³ cells per well. In a dilution plate, pooled mouse serum was diluted in series with a final volume of 12.5 μL. Then 12.5 μL of SARS-CoV-2 was added to the dilution plate at a concentration of 1.2 × 10⁴ pfu/mL.
After 1 h incubation, the media remaining on the 384-well plate was removed and 25 μL of the virus/serum mixture was added to the 384-well plate. The plate was incubated for 20 h after which the plate was fixed for 1 h. Each well was then washed three times with 100 μL of 1xPBS 0.05% tween. 12.5 μL of human polyclonal sera diluted 1:500 in Perm/Wash buffer (BD Biosciences 554723) were added to each well in the plate and incubated at RT for 2 h. Each well was further washed three times and peroxidase goat anti-human Fab (Jackson Scientific) was diluted 1:200 in Perm/Wash buffer, then added to the plate and incubated at RT for 2 h. The plate was then washed three times and 12.5 μL of Perm/Wash buffer was added to the plate then incubated at RT for 5 min. The Perm/Wash buffer was removed and TrueBlue peroxidase substrate was immediately added (Sera Care 5510–0030). Sera were tested in triplicate wells. Normal human plasma was used as negative controls for serum screening.

For plaque assays corresponding to titers presented in S1C Fig, plaque assays were carried out as described in Hanley et al. (2010) with the exception that A549 cells were used with overlay medium of F-12K media supplemented with 10% FBS, pen-strep, and 0.8% methylcellulose [58 (link)].To determine virus titer and for the plaque assays presented in S8C Fig, HEp-2 cells were plated in 24 well plates. When cells were confluent, virus was added as an inoculum for 1 h at 37°C. Overlay medium of DMEM, FBS, pen-strep, and avicel (FMC Biopolymer) was then added on top of inoculum, and cells were incubated for 6 days at 37°C before fixation with 4% paraformaldehyde (Electron Microscopy Sciences) for 10 minutes at room temperature. Cells were then blocked for 30 minutes at 37°C with 5% BSA (Sigma Aldrich). Cells were stained with an antibody specific for RSV F protein for 30 minutes at 37°C, followed by an anti-human horse radish peroxidase secondary antibody (Jackson Immunoresearch, 1:250 dilution) for 30 minutes at 37°C. Finally, cells were incubated with TrueBlue peroxidase substrate (SeraCare) for 10 minutes at RT. Plaques were then counted to determine titer. Biological replicates were performed, where n = 4.

The FRNT₅₀ assay for ZIKV was performed on NHP serum samples. Briefly, fourfold serially diluted serum samples and ZIKV (strain PRVABC59) (≈100 pfu/ml) were incubated for 1 h at 37 °C and added to Vero cells, with overlay medium added after 1 h. After overnight culture, the 96-well plates were stained with an anti-flavivirus monoclonal antibody (MAB10216, Millipore, Burlington, MA USA) and goat anti-mouse IgG (H + L) horseradish peroxidase (HRP)-conjugated secondary antibody (5220–0341, SeraCare Life Sciences, Milford, MA, USA). TrueBlue Peroxidase Substrate (5510–0030, SeraCare Life Sciences, Milford, MA, USA) was added to the plate and spots counted by CTL Biospot Analyzer and Biospot software (Cellular Technology, Ltd, Cleveland, OH, USA). FRNT₅₀ titres were calculated by interpolation between the two points that spanned 50% neutralisation.

Madin-Darby canine kidney (MDCK) cells acquired from the American Type Culture Collection were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated FBS, 1% penicillin/streptomycin, 1% l-glutamine, and 1% sodium pyruvate. MDCKs (4 × 10⁴ cells per well) in a 96-well flat-bottom plate were incubated with lung tissue homogenates for 1 hour at 37°C/10% CO₂. A carboxymethylcellulose (CMC) overlay, generated by mixing 1:1 2× DMEM with 6.4% (w/v) CMC salt (Sigma-Aldrich) diluted in distilled water, was then added to restrict viral spread. Plates were incubated overnight at 37°C/10% CO₂. The overlay was then removed; the cells were fixed in 80% (v/v) of acetone at 4°C, blocked in 5% skim milk powder diluted in 0.05% PBS tween before being stained with mouse anti-influenza A virus NP (CSL Pty Ltd.) followed by secondary anti–mouse-horse radish peroxidase. TrueBlue Peroxidase Substrate (SeraCare) was added, and the wells were imaged using a CTL ImmunoSpot analyzer.