Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells

There is a constant and growing interest in exploiting adenoviruses as vectors for gene therapy when transient expression of a therapeutic protein is necessary. The requirement for an increased viral titre has prompted a search for techniques by which this virus may be assayed with greater speed and simplicity. Conventional plaque assay for quantification of adenoviral vectors titre in current use is laborious and time-consuming (up to 14 days). We report herein a method for the monitoring of adenovirus expressing green fluorescent protein that incorporates rapid and easy sample handling by means of flow cytometric analysis. Cells (HEK293) were infected with adenovirus at various multiplicity of infection (MOI), harvested 17 to 20 h post infection and analysed by flow cytometry. Assumptions were made that one fluorescent cell was infected by a single infectious particle at a relatively low MOI. The adenoviral titre was subsequently estimated from cell analysis in a relatively short time. The results obtained with an E1-complementing cell line (HEK293) were compared with that obtained using a non-complementing cell line (A549). A Poisson distribution successfully modelled the profile of infection as a function of MOI. This provided a better understanding of adenoviral infection at the earliest stage possible. Monitoring of GFP fluorescence and virus propagation in a batch culture of infected cells was subsequently used as a practical application of the validated method.