Real time PCR for rapid determination of susceptibility of adenovirus to antiviral drugs

Abstract

Human adenoviruses (HAdV) are associated with respiratory, ocular and gastrointestinal infections as well as potentially fatal disseminated disease in highly immunocompromised patients. Although there is no specific FDA approved treatment for HAdV infections, some antivirals are used in certain patients. The in vitro antiviral assays for HAdV are not standardized and are usually time consuming. The objective of this study was to evaluate a real time PCR assay for rapid determination of susceptibility of HAdV to antiviral drugs. The nucleoside analogue stavudine (d4T) was used as test drug in A549 cells infected with HAdV5. The antiviral assay measured the reduction of the HAdV DNA levels in culture supernatants by real time PCR using specific primers that amplify a conserved region of the hexon gene. This real time PCR assay demonstrated that stavudine was a selective inhibitor for HAdV5, since the effective concentration 50% (EC50) ranged from 0.08 to 0.12 mM at multiplicity of infection between 0.001 and 1. Furthermore, EC50 showed a high correlation with plaque reduction and virus yield inhibition assays (r2 = 0.9938 and r2 = 0.9468, respectively). In conclusion, the real time PCR-based antiviral assay is rapid, reproducible and could replace classical and more labor-intensive infectivity assays.