A robust cell-based infection model for Rhinovirus C research and antiviral drug discovery

Figures

• 

  Fig. 1. Generation of a stable RV-C15 reporter virus and phenotypic screening of permissive clones. a Schematic of HeLa-CDHR3C529Y generation and workflow of phenotypic clone screening. b Schematic of RV-C15a-mGL virus genome orientation. Created in BioRender. Lyoo, E. (2026) https://BioRender.com/oydff8k. c Primary screening of 80 monoclonal clones infected with RV-C15a-mGL. Infection efficiency was quantified at 3 dpi by HCI as the percentage of mGL-positive cells. The red dashed line indicates 50% infection threshold. Each circle represents an individual replicate (minimum n = 3). Replicates with infection rates > 50% are highlighted in green circles, and replicates with ≤50% infection are shown in gray circles. Selected clones for further evaluation are indicated by colored bars and labels (green = permissive, red = negative). d Flow cytometry analysis of surface CDHR3 expression in permissive and non-permissive clones. Infection efficiency is shown as green circles (% mGL-positive cells), and CDHR3 expression as red triangles (mean fluorescence intensity; MFI). Each point represents the mean of two technical replicates from an independent experiment. Colored bars indicate the…

• 

  Fig. 2. Development and validation of a 96-well RV-C antiviral assay. a Infection kinetics of RV-C15a-mGL in HeLa-CDHR3C529Y mono29 cells at different MOIs and time points. Data are presented as mean ± SD (n = 3). Selected MOI for antiviral assay is highlighted in green. b Pilot RV-C15a-mGL antiviral assay with reference compounds BF738735, Pirodavir, and Rupintrivir. Compounds were tested in 8-point dose-response assays starting at 1 µM. Assay plates were fixed at 3 dpi and analyzed by HCI. One representative plate is shown. Each point on the inhibition curves represents an individual replicate. Two operators performed the assay independently (total n = 4). VC, virus control. CC, cell control. c Virus yield reduction assay with Rupintrivir and Vapendavir. Viral RNA in supernatants was quantified at 72 hpi. Data are mean ± SD (n = 3). d, e Proof-of-concept inter-operator assay reproducibility across >300 plates containing pre-spotted reference compounds in assays with RV-A16, RV-B14, and RV-C15a-mGL. f Representative images of RV-C11a-mGL and RV-C41a-mGL infection and antiviral assays with Rupintrivir (n = 3).

• 

  Fig. 3. Miniaturization of the RV-C15a-mGL assay to 384-well format. a Schematic of 384-well format antiviral assay procedure. Created in BioRender. Lyoo, E. (2026) https://BioRender.com/oydff8k. b Assay robustness in 384-well format across RV-A16, RV-B14, and RV-C15a-mGL assays using >100 pre-spotted plates with reference compounds. c Distribution of pIC50 values (−log10 IC50, M) for BF738735 and Rupintrivir in the RV-C15a-mGL assay. Each dot represents an individual run; boxes indicate median, interquartile range (IQR), and whiskers (1.5× IQR). d Comparison of IC50 values for Pirodavir, Rupintrivir, and BF738735 in the RV-A16, RV-B14, and RV-C15a-mGL assays between the 96-well and 384-well formats.

• 

  Fig. 4. Preliminary HTS of a 10,240-compound library. a Workflow of RV-C15a-mGL HTS campaign in 384-well format. b Scatterplot summarizing primary screening at 10 µM and three reference compounds BF738735, Pirodavir, and Rupintrivir (yellow). Compounds with inhibition > 60% and viability > 90% are highlighted in green. c Assay performance across 32 HTS plates. VC and CC are plotted against the left Y-axis in % infection, while Z′-factor is plotted against the right Y-axis. VC, virus control. CC cell control. d Inhibition and viability curves for five top hit compounds from mini dose-response assays (0.2–25 µM, n = 4). e Distribution of IC50, CC50, and selective index (SI = CC50/IC50) values for 19 confirmed hits from mini dose-response assays. Five top hits (SI > 7) are highlighted in green.