In this study, we describe the use of nuclease-resistant molecular beacons (MBs) for the real-time detection of coxsackievirus B6 replication in living Buffalo green monkey kidney (BGMK) cells via Tat peptide delivery. A nuclease-resistant MB containing 2-Omethyl RNA bases with phosphorothioate internucleotide linkages was designed to specifically target an 18-bp 5 noncoding region of the viral genome. For intracellular delivery, a cell-penetrating Tat peptide was conjugated to the MB by using a thiol-maleimide linkage. Presence of the Tat peptide enabled nearly 100% intracellular delivery within 15 min. When the conjugate was introduced into BGMK cell monolayers infected with coxsackievirus B6, a discernible fluorescence was observed at 30 min after infection, and as few as 1 infectious viral particle could be detected within 2 h. The stability and the intracellular delivery properties of the modified MBs enabled real-time monitoring of the cell-to-cell spreading of viral infection. These results suggest that the Tatmodified, nuclease-resistant MBs may be powerful tools for improving our understanding of the dynamic behavior of viral replication and for therapeutic studies of antiviral treatments.detection ͉ infectious diseases ͉ real time

Here we describe a new hybrid fluorescent nanoprobe composed of a nuclease-resistant molecular beacon (MB) backbone, CdSe-ZnS core-shell quantum dots (QDs) as donors, and gold nanoparticles (Au NPs) as quenchers, for the real-time visualization of virus replication in living cells. By using a Au NP-MB to QD ratio of 6 : 1, a 7.3-fold increase in fluorescent signal was achieved upon target binding. For living cell experiments, a hexahistidine-appended Tat peptide was self-assembled onto the QD surface to provide nearly 100% non-invasive delivery of the QD-MB-Au NP probes within 2 h. By directly visualizing the fluorescent complexes formed with the newly synthesized viral RNA, this QD-MB-Au NP probe provided sensitive and real-time detection of infectious viruses as well as the real-time visualization of cell-to-cell virus spreading.

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Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5 noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays.Hepatitis A virus (HAV), a positive single-stranded RNA virus, was previously classified as enterovirus type 72 (14) and has now been classified in the Hepatovirus genus of the Picornaviridae family (15). These single-plus-stranded RNA viruses can directly translate plus-strand RNA genomes into protein using the host ribosomes. The plus strand is transported to the infected cell via specific membrane vesicles, where it is copied into full-length minus strands. These minus strands then serve as templates for the synthesis of plus-strand genomic RNA molecules (1, 2).HAV is known to cause acute liver infection with a discrete onset of symptoms (e.g., fever, malaise, and nausea), followed in several days by jaundice. Person-to-person transmission through the fecal-oral route is the primary means of HAV transmission. Outbreaks and periodic cases also occur from exposure to fecally contaminated food or water (Centers for Disease Control and Prevention). Most picornaviruses are lytic, causing distinctive cytopathic effects and replicating in an 8-h cycle under one-step growth conditions. The growth of HAV in cell culture systems, however, is much slower and nonlytic and does not produce a detectable cytopathic effect in infected cells (13). Cromeans et al. reported the isolation of a cytopathic HAV variant from the rapidly replicating isolate HM-175 that is lytic for fetal rhesus monkey kidney (FRhK-4) cells (4). However, complete lysis still did not occur until 5 to 6 days postinfection (p.i.).Conventional methods for detecting infectious HAV rely on viral propagation in cell culture, radioimmunoassay, immunofluorescence, or plaque assay; these methods are difficult to perform, and it may take weeks before the viruses reach sufficiently high amo...

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