Biospot software

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.

Focus reduction neutralization tests (FRNTs) were used to determine neutralization activities as an additional platform aside from the plaque assay. Serial dilutions of serum starting at a final concentration of 1:20 were mixed with 10³ focus-forming units of virus per well and incubated for 1 hour at 37 °C. A pooled prepandemic serum sample served as a control. The antibody-virus mixture was inoculated onto Vero E6/TMPRSS2 cells (JCRB) in 96-well plates and incubated for 1 hour at 37 °C. An equal volume of methylcellulose solution was added to each well. The cells were incubated for 16 hours at 37°C and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse mAb against SARS-CoV-1/2 nucleoprotein [clone 1C7C7 (Sigma-Aldrich)], followed by a HRP-labeled goat anti-mouse immunoglobulin (Sigma-Aldrich; A8924). The infected cells were stained with TrueBlue Substrate (SeraCare Life Sciences) and then washed with distilled water. After drying, the focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology). The IC₅₀ was calculated from the interpolated value from the log(inhibitor) versus normalized response, using variable slope (4 parameters) nonlinear regression performed in GraphPad Prism (version 9.0).

Antiviral susceptibilities were determined by using a focus reduction assay as previously reported^{5 (link)–7 (link)}. Briefly, VeroE6-TMPRSS2-T2A-ACE2 cells in 96-well plates were infected with the indicated virus at 100–400 focus forming unit/well. After incubation at 37 °C for 1 h, the inoculum was replaced with 1% Methyl Cellulose 400 (FUJIFILM Wako Pure Chemical Corporation) in culture medium containing serial dilutions of the antiviral compounds. The cells were incubated for 18 h at 37 °C and then fixed with formalin. The cells were stained with a mouse monoclonal antibody against SARS-CoV-2 nucleoprotein, clone N45 (TAUNS Laboratories, Inc., Japan), followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories Inc.). Foci were visualized by using TrueBlue Substrate (SeraCare Life Sciences). The focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology). The 50% inhibitory concentration (IC₅₀) values and 95% confidence intervals were calculated by using GraphPad Prism 9.3.0 (GraphPad Software).

Neutralization activities of SARS-CoV-2 were determined by using an FRNT as previously described^{14 (link)}. Serial dilutions of plasma were mixed with 1,000 focus-forming units of virus/well and incubated for 1 h at 37 °C. The antibody-virus mixture was inoculated on VeroE6/TMPRSS2 cells in 96-well plates in duplicate and incubated for 1 h at 37 °C. An equal volume of 1.2% Avicel RC-581 (DuPont Nutrition USA) in culture medium was added to each well. The cells were incubated for 24 h at 37 °C and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse monoclonal antibody against SARS-CoV-1/2 nucleoprotein [clone 1C7C7 (Sigma-Aldrich)], followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (SeraCare Life Sciences). The infected cells were stained with TrueBlue Substrate (SeraCare Life Sciences) and then washed with distilled water. After cell drying, the focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology). The results are expressed as the 50% focus reduction neutralization titer (FRNT₅₀). The FRNT₅₀ values were calculated by using GraphPad Prism (GraphPad Software).