Development of a rapid fluorescence-based adenovirus : inactivation assay

Abstract

Screening of potential antiviral product prototypes is typically performed using The American Society for Testing and Materials (ASTM) method E1052 “Efficacy of Antimicrobial Agents against Viruses in Suspension.” Adenovirus (ADV) is a recommended test virus and demonstrates intermediate resistance to chemical inactivation. The procedures commonly employed to quantify infectious virus are median cell culture infective dose (CCID50) and the plaque assay (PA). The rate-limiting step in both of these procedures is the time needed to form observable cytopathic effect (CPE) in infected cells, which takes approximately 7 days for ADV. Virus inactivation studies were performed according to ASTM E1052 using the traditional PA method as well as an experimental fluorescence-based method utilizing a recombinant ADV (ADV-GFP) that cconstitutively expresses green fluorescent protein (GFP). Infective units were determined by counting the number of fluorescent green cells or plaques. The log 10reduction (LR) values of virus inactivation were determined for several different concentrations of ethanol and hypochlorite using the traditional PA method and the ADV-GFP method. The LR values using the ADV-GFP method were similar, within +/- 0.5 log 10, to the LRvalues obtained using the traditional PA method. The ADV-GFP method was shown to be an acceptable substitute yielding results in 2 days vs. 7 days for the traditional methods. In addition, A549 cells were stably transfected with a plasmid construct containing a GFP gene under control of the ADV early gene E2 promoter for the eventual use in a newly conceived assay for quantitating ADV infection.