Huh-7 monolayers treated with CH223191 or kynurenine and infected with JUNV as mentioned above were fixed at 48 h pi with methanol for 10 min at −20 °C, washed thrice with PBS, and stored in fresh PBS at 4 °C until processing. The following antibodies were used: primary mouse monoclonal anti-NP antibodies (1:200) (NA05-AG12) [21 (link)] and secondary Alexa Fluor 488 conjugated anti-mouse goat antibodies (1:200) (A-11001) (ThermoFisher Scientific, Waltham, MA, USA). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1:1000) (Sigma-Aldrich, St. Louis, MO, USA). Microscopy and photography data were obtained using an Olympus IX71 fluorescence microscope with a QImaging Exi-Aqua camera attached (pixel size: 6.45 μm; photodetector area: 1392 × 1040 pixels2). Quantification of fluorescence data was performed using Fiji software (version 1.53v) (National Institutes of Health, Bethesda, MD, USA). Manual quantifications of DAPI-stained nucleus and NP-positive cells were performed manually through the cell counter plugin included in the Fiji software (version 1.53v). Cells were considered positive if NP viral antigen staining could be detected. Percentage of NP-positive cells was expressed as the ratio between NP-positive cells and total cells in each field and then the average percentage was determined and presented. Fluorescence intensity of NP-positive cells was obtained by measuring mean gray value of individual cells and subtracting background fluorescence. Viral foci were delimited by considering groups of NP-positive cells surrounded by NP-negative cells. Viral foci were counted on 3 different images from 3 different randomly chosen fields and the average foci size was determined by the number of cells that formed them.
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