UC Riverside

Viruses pose a serious threat to public health and safety. Rapid and efficient detection of viruses is crucial for the prevention of disease spread and timely clinical management. Traditional methods for detection of viral infection that rely on viral isolation and culture techniques continue to be the golden standards used for detection of infectious viral particles. However, improved methods for rapid and reliable detection and quantification of viruses are required for public health assessment. New techniques relying on visualization of live cells can shed some light on understanding virus-host interaction for early stage detection and potential drug discovery. One such technology is the use of Molecular beacons (MBs), which produce fluorescence upon target binding and offer a simple, separation free scheme for sensitive detection of infectious viruses. In this study, we developed several FRET (fluorescence resonance energy transfer)-based MBs combined with fluorescence microscopy to directly visualize the fluorescent hybrids with viral RNA as an indication of viral infection. To prevent nucleolytic degradation, MBs were designed containing 2'-O-methyl RNA bases with phosphorothiote linkages and a cell-penetrating Tat peptide was attached to facilitate non-invasive intracellular delivery. To demonstrate the flexibility of this technique to be adapted into a high-throughput screening method, a flow cytometry (FC) based system was utilized and the simplicity of using a flow based scheme for rapid identification of virus infected cells was demonstrated. Confluent cell monolayers were infected with virus dilutions followed by incubation with MB probes and fluorescent intensity was monitored using fluorescence microscope as well as flow cytometry. The sensitivity of the assay to detect less than 1% infected cells in a mixed cell population was shown. The illumination of fluorescent cells increased in a dose-responsive manner and enabled direct quantification of infectious viral doses. Fluorescent signal from virus infected cells was discernible as early as 15 min thereby enabling rapid detection. The specificity of the MBs to differentiate between different subtypes of viruses was also demonstrated. The specific nature of these probes enable their utility for rapid diagnosis of viral infection and provides an better understanding of the multiple steps involved in the viral infection process, which will be valuable for the prevention and control of disease.

Another approach to detect the presence of infectious viruses was explored by probing the viral protease activity in vivo. Proteases are involved in many essential cellular processes and are used as the key virulence factors for pathogenic infection. These properties make proteases a prime target for detailed investigation to better understand the disease development process and to identify targets for drug treatment. The approach was to generate a quantum dot (QD)-modified, protease-specific protein module that can be utilized as a FRET based nano-biosensor for probing viral protease activity in vivo. The site-specific incorporation of an acceptor fluorophore was accomplished using a cysteine residue and conjugation of QDs was facilitated by the presence of a hexa-histidine tag. Simple purification of the QD-FRET substrate was achieved by the presence of an elastin like protein (ELP) and intracellular delivery of the substrates was enabled by the use of a flanking Tat peptide. The effectiveness of the FRET substrate to probe intracellular viral protease activity was studied by measuring the whole-cell fluorescence ratio between the QD and the acceptor fluorophore. The utility of the assay system was validated for high-throughput screening of viral protease inhibitors.