E80i microscope

For IF, cell monolayers were prepared in Millicell EZ 4-well chamber slides (Merck) and fixed in PBS with 4% paraformaldehyde. Expression of EV protein antigens was tested by indirect IF using multiple pan-EV mAbs (ESM Table 1) directed to: (1) the VP1 capsid protein (mAbs 9D5, 6-E9/2, 5D8/1 [34 (link)]); (2) the 3D viral RNA polymerase (mAbs 3D-02 and 3D-05—our laboratory). Further controls included stainings with mAbs directed to coxsackieviruses group B; echoviruses 4, 6, 9, 11, 30, 34; echoviruses 4, 6, 9, 11, 30; and polioviruses 1–3. Alexa Fluor 488-goat anti-mouse IgG served as secondary antibody. Slides were counterstained with Evans blue. VP1 staining was deemed positive when granular cytoplasmic fluorescence was detected in infected cells. Staining for 3D RNA polymerase (3Dpol) typically produced speckled fluorescence in the nuclear area of persistently infected cells vs cytoplasmic staining of acutely infected cells. Images were taken with a Nikon E80i microscope and adjusted using Adobe Photoshop.

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.