Immunofluorescence Assay for EV Protein Detection

Cell monolayers were prepared in Millicell EZ 4-well glass slides (Merck, Darmstadt, Germany) and fixed in PBS containing 4% paraformaldehyde. Expression of EV protein antigens was evaluated by immunofluorescence with anti-EV monoclonal antibodies (Table S1) targeting either the VP1 capsid protein (mAbs 9D5, 6-E9/2, 5D-8.1) [21 (link)] or the viral 3D RNA polymerase (mAb 3D-02 and 3D-05 from our own laboratory). For additional virus typing, select monolayers were also stained with mAbs specific for group B coxsackieviruses; echoviruses 4, 6, 9, 11, 30, 34; polioviruses 1–3 (Table S1). Alexa Fluor 488-goat anti-mouse IgG (ThermoFisher) was used as secondary antibody. The slides were counterstained with Blue Evans. VP1 staining was deemed positive if fine granular cytoplasmic fluorescence was detected in infected cells. In persistently infected cells, the 3Dpol staining typically produced diffuse speckled fluorescence in the nuclear area. The images were taken with a Nikon E80i microscope and adjusted in brightness and contrast using Adobe Photoshop.