Rapid cell culture-based assay for the detection of infectious viruses and viral vectors

The determination of infectious titers of samples containing virus particles is critical for clinical gene therapy applications. Currently available methods that determine the infectivity of viruses/viral vectors are time-consuming. In this study, we developed cell culture-based infection assays that rapidly detect wide ranges of DNA/RNA viruses/viral vectors. Viruses analyzed in this study included Adenovirus type 5, Adenovirus type 41, Vaccinia virus, Measles virus, Coxsackie virus B5, Respiratory Syncytial Virus, and Adenovirus-, Adeno-associated virus-, Retrovirus-, and Lentivirus-based vectors. Susceptible cells were infected for 3 h with samples containing infectious viruses/viral vectors. In developed assays, differences in Cq values (∆Cq) were measured between cells incubated with infectious virus for 3 h and the two controls, including 0 h and heat-inactivated controls. Controls were included to detect background signals. An infection assay with a ∆Cq greater than 3.3 was considered positive. The reproducible and repeatable infection assays were functioning based on virus entry into the cells. For all tested viruses/viral vectors, ∆Cq above 3.3 was detected between cells incubated with infectious virus for 3 h and at least one control, indicating the positive functioning of the assay. The 0 h and heat-inactivated controls that were crucial for the estimation of the background, were successfully applied to all viruses/viral vectors tested in this study. We have developed a rapid cell-culture-based assay for detection of infectious viruses. The assay could be applied to all tested viruses/viral vectors, and has the potential to become a valuable tool in clinical virology and infectious virus diagnostics.