Modified Vaccinia Virus Ankara Titration Using Crystal Violet- or Immuno-Staining in DF-1 Cells

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an outstanding safety profile. The gold standard method to titrate MVA is to immune stain plaques formed in MVA-infected monolayer of primary chicken embryo fibroblasts. Recently, DF-1, an immortal chicken embryo fibroblast line, has also been used. We recently found that MVA plaques in infected DF-1 monolayer could be readily visualized using crystal violet staining, greatly reducing the time and cost of the MVA titration by plaque assay, and with comparable titration results to the gold standard method using immunostaining. Here we present the protocols for MVA amplification and titration using crystal violet- and immuno-staining in DF-1 cells, respectively. Briefly, a fully confluent monolayer of DF-1 cells is infected with MVA at a series of dilutions. The cells are overlaid with media containing methylcellulose, allowing the formation of distinct plaques. After incubating for 2-3 days, the cells are stained with crystal violet or vaccinia virus antiserum, followed by plaque counting.