2.3. Plaque-Forming-Assay

Plaque assay was performed to quantify infectious viral particles. Vero cells were seeded in 6-well plates (3 × 10⁵ per well) and infected with 100 µl of serial dilutions from cell culture supernatants. After 2 h, the medium was removed and the cells were layered with 0.4% agarose solution (SeaPlaque^® Agarose; Lonza, Basel, Switzerland). Five days after infection, the agarose overlay was removed and cells were fixed with 4% formaldehyde in PBS for 20 min. Visualization of the plaques was achieved by staining with crystal violet (0.1% in 20% ethanol; Merck, Darmstadt, Germany) 15 min at room temperature. After washing with distilled water to remove excess crystal violet solution, the plates were dried, and plaques were counted.