Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Traditional double-strand RNA detection assay usually is not sensitive enough to detect double-strand RNA formed in negative-sense RNA virus infection.Therefore the interaction between double-stranded RNA and host pattern recognition receptors has remained largely elusive for these viruses.Utilizing this protocol we are able to visualize and characterize the interaction between viral nuclear protein, double-strand RNA and host a PRRs at a single-cell level for negative-sense RNA virus.This is a multiplex assay that allows for simultaneous staining of double-stranded RNA and two viral or host protein targets in individual cells.Unlike the RNA-FISH assay this technique is RNA sequence independent and usually compatible with antibody detection of protein targets.Begin 24 hours prior to infection by seeding 200, 000 human lung epithelial A549 cells onto Poly-D-Lysine coated glass cover slips in 12-well plates and growing them at 37