Adaptation of the plaque assay methodology for dengue virus infected HepG2 cells

The HepG2 cell line is a useful tool for studying dengue virus-cell interactions but as it grows in clumps rather than monolayers, it does not readily adapt itself to the standard plaque assay technique. We therefore sought to develop an indirect plaque assay methodology. Initially HepG2 cells were infected with dengue virus serotype 2 and post-infection incubated for between 0 and 16 h before being treated with trypsin to separate the cells, followed by dilution and plating onto pre-grown monolayers of Vero cells in six well plates. After 7 days incubation and crystal violet staining, plaques were observed at all time points, although there was a relationship between number of plaques and post-infection incubation time, with the longest post-infection incubation time giving the highest number of plaques. To validate the assay with respect to virus input, the experiment was repeated at both the 0 and 16 h post-infection incubation times with different virus: cell levels. At both post-infection incubation times the response of input virus to plaque number was linear. This is a useful adaptation of the plaque assay methodology and one that may be applicable to other virus/cell line combinations.