Improved methods for rapid and reliable detection of infectious viruses are needed to enable rapid and quantitative determination of their presence for public health assessments. Molecular beacons (MB) provide a label-based and separation-free detection scheme that produces fluorescence upon target binding. By directly visualizing the fluorescent hybrids with newly synthesized viral RNA, the combined cell culture – molecular beacon assay provides rapid and sensitive detection of infectious viruses. For real-time studies in living cells, however, the durability of MBs is affected due to cellular nuclease degradation. The current method requires cell fixation and permeabilization to denature and crosslink the proteins to maintain cellular architecture and to protect the injected MB structures. It is essential to improve the efficacy of MBs to probe and quantify infectious viruses for in vivo studies. In this report, we developed nuclease-resistant MBs for the detection of coxsackievirus B6 Schmitt strain in buffalo green monkey kidney (BGMK) cells via TAT peptide delivery. A MB, CVB6, specifically targeting the 18-bp 5' noncoding region of the viral genome, was designed and synthesized. The MB structure was modified by combining 2'-O-methyl RNA bases with phosphorothioate internucleotide linkages to allow MB CVB6 to be resistant to cleavage by DNase I, and the hybrids with viral RNA to become refractory to digestion by RNase H. Cell-penetrating TAT peptides were conjugated to MB CVB6 using thiol-maleimide linkages to introduce MB CVB6 into living cells without permeabilization. At 5 µM, MB CVB6 demonstrated self-delivery within 20 minutes with nearly 100% efficiency. Confluent BGMK monolayers were infected with 10-fold serial virus dilutions and discernible fluorescence was observed around 30 minutes postinfection, suggesting that the entry and uncoating of virions occurred within 30 minutes after infecting the cells. The degree of fluorescence was monitored and it was observed that each viral reproductive cycle was completed between 10 and 12 hour postinfection. The number of fluorescent cells increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages. The validation of the fluorescence assay for viral quantification was also demonstrated by comparing with traditional plaque assays.

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