Journal: bioRxiv

Article Title: Validation of Flavivirus Infectious Clones Carrying Fluorescent Markers for Antiviral Drug Screening and Replication Studies

doi: 10.1101/2023.04.03.535319

Figure Lengend Snippet: ( A ) Schematic representation of the experimental protocol used to perform marker stability assay. Vero cells were infected with P 0 stocks of recombinant ZIKV, KUNV, DENV2 or DENV4 with mCherry, oxGFP or NLuc markers and incubated until development of CPE or visible marker expression. P1 stocks were collected and used to infect Vero cells for the next passage until passage 4 (P1 to P4 stocks). Viral stocks were titrated by FFA using a mouse pan-flavivirus anti-Env antibody (Mab 4G2). mCherry signal was measured by quantification of fluorescence using a Cellomics ArrayScan VTI microscope. oxGFP expression was detected by immunofluorescence assay using an anti-GFP antibody. For the measurement of NanoLuciferase activity, a second 96-well plate was infected with P1 to P4 stocks according to the end-point titration method and NLuc was measured with the Nano-Glo Luciferase Assay System after cell lysis, on a microplate reader. ( B ) Evolution of the transgene expression through serial passages for each virus. The data represent the expression of the marker reported to the infectious titer. All experiments were performed in triplicate and results are represented as mean +/-SD.

Article Snippet: For cell fluorescence counting, 9,000 cells per well were cultured in black opaque 96-well Microplate (PerkinElmer), infected with virus stock at an MOI 0.01 (ZIKV-mCherry) or MOI 0.05 (DENV2-Ct-mCherry) and prepared for imaging at 72 h post infection.

Techniques: Marker, Stability Assay, Infection, Recombinant, Incubation, Expressing, Fluorescence, Microscopy, Immunofluorescence, Activity Assay, Titration, Luciferase, Lysis, Virus