Visualizing Trypsin-Activated Rotavirus Infection Using a Plaque Assay

Begin with a diluted recombinant rotavirus suspension pre-treated with trypsin.
Trypsin cleaves outer capsid proteins, activating the viruses for efficient attachment.
Add the diluted virus suspension onto a monolayer of permissive epithelial cells and incubate.
Gently shake the plate at regular intervals to evenly distribute the viruses.
The virus enters, replicates, and forms new viruses. Aspirate the suspension to remove extracellular viruses.
Overlay the cells with a semi-solid agarose medium containing trypsin. Allow the agarose to solidify, which restricts virus spread, and incubate.
Infected cells lyse, releasing viruses and forming clear zones called plaques. Trypsin activates viruses and supports reinfection, increasing plaque formation.
Add another overlay containing a cell-permeable dye and incubate in the dark.
The dye stains viable cells red, while the plaques remain unstained.
Using a light box, visualize the plaques.
Extract the recombinant viruses from the plaques and transfer them into fresh media for downstream analysis.
Add trypsin to 100 microliters of clarified cell lysate to a final concentration of 10 micrograms per milliliter and incubate the mixture at 37 degrees Cels