Figure 1

MabJ2 detects viral replication-induced dsRNA in high content image based assays. (A) Cells with dsRNA replication centers are positive for newly synthesized viral protein. HeLa cells were infected with the indicated HRV or CV serotypes, fixed and stained with mabJ2 (red) or capsid specific antibodies (green). CVB3, CVB4, HRV37 and HRV14 were stained with a rabbit polyclonal serum (rpc); HRV1A and 16 were stained by mabR16-7 covalently labelled with Alexa488 (R16-7-488). Magnification 60×; scale bar 20 μm. (B) Appearance of dsRNA replication centers is moi dependent. Example overview of a 96 multiwell plate of HeLa cells infected with serial dilutions of indicated HRV or CV serotypes. Imaging by automated microscopy was with 10× magnification. One out of nine images per well is shown for each condition. dsRNA replication centers (green) and DAPI stained nuclei (blue) are shown. Scale bar 100 μm. (C) An example for automated fluorescence image analysis to score infection of HeLa cells with HRV16 (moi 0.3) with raw images on the left and an image processed and pseudocolored with a Matlab algorithm on the right side. Scale bar 100 μm. (D) Example for the quantification of moi dependent fraction of infected cells (infection index) of the experiment shown in (B), and analysis by the scoring algorithm presented in (C). More detailed characterisations (time, dose) of this assay are shown in the subsequent figures.