Reliable and fast viral detection and quantification protocols are a requirement for the advance of basic research and clinical approaches with wild type or recombinant viruses. However， available cell-based assays are either time-consuming or require labeled viral particles， which may alter virus biology or pose safety issues in clinical applications. Since adenoviruses constitute a major healthcare burden but also， when engineered， widely used vectors in vaccination and gene and oncolytic therapies， herein we developed a genetically encoded switch-on fluorescent biosensor consisting of a cyclized Green fluorescent protein-cVisensor-with an adenoviral protease cleavable site as a switch. After initia... More
Reliable and fast viral detection and quantification protocols are a requirement for the advance of basic research and clinical approaches with wild type or recombinant viruses. However， available cell-based assays are either time-consuming or require labeled viral particles， which may alter virus biology or pose safety issues in clinical applications. Since adenoviruses constitute a major healthcare burden but also， when engineered， widely used vectors in vaccination and gene and oncolytic therapies， herein we developed a genetically encoded switch-on fluorescent biosensor consisting of a cyclized Green fluorescent protein-cVisensor-with an adenoviral protease cleavable site as a switch. After initial sensor optimization (35% increase in performance)， whole-cell biosensors were established-by stably expressing cVisensor in mammalian cells-and used for live-cell monitoring of adenovirus infection as the intracellular biosensor is specifically activated by the viral protease. A rapid flow cytometry-based bioassay using cVisensor cells was established 48 h postinfection， showing an estimated limit of detection of 10 infectious particles/mL， in-line with previously reported flow cytometry assays requiring labeled virus， and significantly faster than standard plaque-forming assays requiring up to 14 days. cVisensor was also successfully applied in the detection of HIV-1 protease activity， validating its wider potential for the detection of other viruses. Overall， this work presents a fast and easy method for detection and quantification of label-free infectious virus， allowing the establishment of new biosensing platforms for basic research in virology and biotechnological applications of recombinant virus biopharmaceuticals.
