Visualization and detection of infectious coxsackievirus replication using a combined cell culture-molecular beacon assay

Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5' noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.