TY - JOUR
T1 - Detection and Quantification of Label-Free Infectious Adenovirus Using a Switch-On Cell-Based Fluorescent Biosensor
AU - Guerreiro, Miguel Ricardo
AU - Freitas, Daniela Filipa
AU - Alves, Paula Marques
AU - Coroadinha, Ana Sofia
PY - 2019/6/28
Y1 - 2019/6/28
N2 - 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 105 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.
AB - 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 105 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.
KW - Cell-based bioassay
KW - Fluorescent biosensor
KW - Intein
KW - Label-free
KW - Protease
KW - Quantification
KW - Virus
UR - http://www.scopus.com/inward/record.url?scp=85067960171&partnerID=8YFLogxK
U2 - 10.1021/acssensors.9b00489
DO - 10.1021/acssensors.9b00489
M3 - Article
C2 - 31117363
AN - SCOPUS:85067960171
VL - 4
SP - 1654
EP - 1661
JO - Sensors
JF - Sensors
SN - 1424-8220
IS - 6
ER -