In the era of highly advanced genetic technologies, we have witnessed a turning point for the development of live viral vaccines. Conventional empirical vaccine development processes are now being replaced by more rational reversegenetics-based ones. With this trend, much attention will be focused on mechanism-based design of less pathogenic and more immunogenic virus stains. Mathematical models for intracellular virus growth can support this design process by providing a tool to systematically analyze the viral infection regulatory network, identify critical regulatory mechanisms or components for redirecting viral phenotypes, and reverse engineer desirable phenotypes. One-step infection of cell monolayers. Cells were harvested, resuspended in growth medium, and plated into six-well plates at a concentration of 5 3 10 5 cells per 2 ml per well. Plated cells were returned to the incubator and allowed to grow overnight. The next day, two representative cell monolayers were harvested and counted to give an approximate number of cells per well. Each monolayer was then incubated with 200 ll of virus inoculum (MOI 3) for 1 h to allow virus adsorption. The plates were rocked gently every 20 min to evenly distribute virions on the monolayers during the adsorption step. After the adsorption period, the monolayers were rinsed twice with 1 ml of HBSS and then placed under 2 ml of infection medium for incubation. Medium samples of 200 ll including virion particles were taken from each well at 2, 3, 4, 6, 8, 10, and 20 h postinoculation. Samples were kept frozen at À90 8C until their analysis by the plaque assay.