Virus Propagation and Cell-Based Colorimetric Quantification

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and potentially Guillain-Barré syndrome in adults. Research on ZIKV to understand the disease mechanisms is important to facilitate vaccine and treatment development. The method of quantifying viruses is crucial and fundamental in the field of virology. The focus forming assay (FFA) is a virus quantification assay that detects the viral antigen with antibodies and identifies the infection foci of cells using the peroxidase immunostaining technique. The current study describes the virus propagation and quantification protocol using both 24-well and 96-well (high throughput) formats. Compared with other similar studies, this protocol has further described foci size optimization, which can serve as a guide to expand the use of this assay for other viruses. Firstly, ZIKV propagation is performed in Vero cells for 3 days. The culture supernatant containing ZIKV is harvested and quantitated using the FFA. Briefly, the virus culture is inoculated onto Vero cells and incubated for 2-3 days. Foci formation is then determined after optimized staining processes, including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. The stained virus foci are visualized using a stereo microscope (manual counting in 24-well format) or software analyzer (automated counting in 96-well format). The FFA provides reproducible, relatively fast results (3-4 days) and is suitable to be used for different viruses, including non-plaque-forming viruses. Subsequently, this protocol is useful for the study of ZIKV infection and could be used to detect other clinically important viruses.