Journal: bioRxiv

Article Title: Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

doi: 10.64898/2026.01.13.699296

Figure Lengend Snippet: A. Comparison of cell toxicity versus viral-GFP intensity (relative fluorescence units) at 7 days post-infection with RSVA or hPIV3 (both at MOI 0.02) with the epithelial cell lines VeroE6, Calu-3 or LLC-MK2 (n=3). Dashed box indicates cell lines chosen for subsequent antiviral assays. B-C. Representative images of the antiviral infection assay in 96-well format demonstrating dynamic range, here showing RSV infection of VeroE6 cells with and without single or dual-drug antiviral treatments. Cytopathology is visualised by endpoint crystal violet staining of adherent cells (B), whilst the fluorescence micrograph of same representative assay plate (C) demonstrates visualisation of GFP-tagged RSVA infection. D. Relationship between cell viability (quantified by crystal violet absorbance) and viral GFP fluorescence, as normalised against virus-only and mock controls. Representative dose response curves are modelled on data for remdesivir against RSVA. E-L. Dose response curves for monotherapy concentrations of remdesivir (E&I), favipiravir (F&J), EIDD-1931 (G&K), and ribavirin (H&L) tested against RSVA (E-H) or hPIV3 (I-L) infection. The viral inhibition % model is shown as the blue line, while cytotoxicity (as a % reduction of adherent cells) is indicated by the red line. The shaded box indicates drug concentrations with cytotoxicity exceeding 20%. (n=5 for RSVA, n=3-4 for hPIV3)

Article Snippet: Human lung adenocarcinoma cell line Calu-3 (HTB-55, ATCC) and rhesus monkey kidney cell line LLC-MK2 (CCL-7, Caltag-Medsystems Ltd) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) and 1× penicillin/streptomycin.

Techniques: Comparison, Fluorescence, Infection, Staining, Virus, Inhibition

Journal: bioRxiv

Article Title: Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

doi: 10.64898/2026.01.13.699296

Figure Lengend Snippet: Dual-combinations of antivirals were assayed for viral inhibitory dose response and cytotoxicity for hPIV3 infection of LLC-MK2 cells. For each panel (A-F), the dose response grid represents % relative viral inhibition and the cytotoxicity response grid indicates % cell viability. Also shown are 3D contour plots of synergy scores as calculated by the HSA model, alongside cytotoxicity at the corresponding antiviral concentrations. (n=3 per combination). Favi = favipiravir, Rem = remdesivir, Mol = EIDD-1931, Riba = ribavirin.

Article Snippet: Human lung adenocarcinoma cell line Calu-3 (HTB-55, ATCC) and rhesus monkey kidney cell line LLC-MK2 (CCL-7, Caltag-Medsystems Ltd) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) and 1× penicillin/streptomycin.

Techniques: Infection, Inhibition

Journal: bioRxiv

Article Title: Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

doi: 10.64898/2026.01.13.699296

Figure Lengend Snippet: A. Graphical representation of methodology for culture of primary airway epithelial cell model at air-liquid interface (ALI) and infection with RSVA or hPIV3. B-C. Representative confocal micrographs of primary airway epithelial cells in ALI culture. Cultures were infected with RSVA, hPIV3, or a mock control and fixed for immunofluorescent staining and imaging at 7 days post infection (dpi). Channels represent signal for DAPI staining of cell nuclei (blue), anti-GFP for the GFP-tagged virus (green), phalloidin for tight junctions (white), alpha-tubulin for ciliated epithelial cells (violet), and MUC5AC to indicate mucus-secretory cells (yellow). Orthogonal views (C) are also shown for representative cultures, with the same immunofluorescent staining. D-G. Cilia-beat frequency (CBF) was analysed using high speed video microscopy at 7 dpi with RSVA (D&F) or hPIV3 (E&G) versus mock. Distribution of measured CBF is shown by histogram and box plot (D-E) for each virus. Representative regions of interest (ROI) from video analysis indicate the relative abundance of detectable cilia movement in mocks versus RSVA (F) and hPIV3 (G) infected cultures. H. Representative well scans showing brightfield and GFP signal at 7 dpi for mock and infection conditions with each virus using cell lines (VeroE6 or LLC-MK2 in 96-well format) compared to primary ALI cultures (24-well format). I. The Ct result from qRT-PCR performed on supernatant (for cell lines) or apical wash (for ALI) samples used for viral genomic sequencing, indicating respective viral load at 7 dpi. J-L. Sequencing results for total mutations (J), intrahost single nucleotide variants (iSNV) and single nucleotide polymorphisms (SNP) (K) for each virus from cell line of ALI culture infection model, and the proportion of mutations which were observed in both models or unique (L). Representative images and functional analyses are shown from triplicate ALI cultures per experimental condition.

Article Snippet: Human lung adenocarcinoma cell line Calu-3 (HTB-55, ATCC) and rhesus monkey kidney cell line LLC-MK2 (CCL-7, Caltag-Medsystems Ltd) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) and 1× penicillin/streptomycin.

Techniques: Infection, Control, Staining, Imaging, Virus, Microscopy, Quantitative RT-PCR, Genomic Sequencing, Sequencing, Functional Assay