Disposable cartridge platform for rapid detection of viral hemorrhagic fever viruses.

Light microscopy is a straightforward and highly portable imaging approach that is used for the detection of parasites, fungi, and bacteria. The detection of individual virus particles has historically not been possible through this approach. Thus, characterization of virus particles is typically performed using high-energy approaches such as electron microscopy. These approaches require purification of virions away from its normal milieu, significant levels of expertise, and only count a small number of particles at a time. To correct these deficiencies we created a platform that allows label-free, point-of-need virus imaging and counting. We adapted a multiplex-capable, interferometric imaging technique to a closed-system that allows real-time particle detection in complex mixtures. To maximize virus particle binding we constructed a disposable device with a constant flow rate of ∼3 µl min-1. Biosafety was achieved by having a sealable sample addition port. Using this platform we were able to readily identify virus binding in a 20 minute experiment. Sensitivity was comparable to laboratory-based assays such as ELISA and plaque assay, and showed equal or better sensitivity against paper-based assays designed for point-of-need use. Our results demonstrate a platform that can be used for rapid multiplexed detection and visualization of whole virus particles. We envision this technology as a sample-to-answer platform for detection and visualization of viruses without the need for prior labeling. This would enable both research investigation of virus particle behavior and morphology and have the potential to be used in a diagnostic context, where direct imaging from samples such as blood and urine would be valuable.

New Approaches for Virus Detection through Multidisciplinary Partnerships.

A critical requirement for controlling outbreaks of viral infection is sensitive and accurate diagnostics, which can be expensive and are frequently located in resource-intensive clinical laboratories. Outbreaks of many viral infections occur in countries where healthcare resources are limited and clinical laboratories scarce. This creates a fulfillment gap, one that could be filled through the development of inexpensive, sensitive, easy to use, and portable diagnostics. Here we describe our efforts to develop a diagnostic technology that detects viruses without needing to label the particle directly. Our approach has the advantage of speed and assay simplicity while maintaining high sensitivity. Essential in this approach has been the assembly of an integrated, diverse, and interdisciplinary team that worked together to evaluate technologies, spin-out a company, and produce a product for infectious disease diagnostics. The synergy of different individuals with complementary skills has been critical for the development of our transformative technology.

Real-Time Capture and Visualization of Individual Viruses in Complex Media.

Label-free imaging of individual viruses and nanoparticles directly in complex solutions is important for virology research and biosensing applications. A successful visualization technique should be rapid, sensitive, and inexpensive, while needing minimal sample preparation or user expertise. Current approaches typically require fluorescent labeling or the use of an electron microscope, which are expensive and time-consuming to use. We have developed an imaging technique for real-time, sensitive, and label-free visualization of viruses and nanoparticles directly in complex solutions such as serum. By combining the advantages of a single-particle reflectance imaging sensor, with microfluidics, we perform real-time digital detection of individual 100 nm vesicular stomatitis viruses as they bind to an antibody microarray. Using this approach, we have shown capture and visualization of a recombinant vesicular stomatitis virus Ebola model (rVSV-ZEBOV) at 100 PFU/mL in undiluted fetal bovine serum in less than 30 min.

An interferometric reflectance imaging sensor for point of care viral diagnostics.

The use of in vitro diagnostic devices is transitioning from the laboratory to the primary care setting to address early disease detection needs. Time critical viral diagnoses are often made without support due to the experimental time required in today's standard tests. Available rapid point of care (POC) viral tests are less reliable, requiring a follow-on confirmatory test before conclusions can be drawn. The development of a reliable POC viral test for the primary care setting would decrease the time for diagnosis leading to a lower chance of transmission and improve recovery. The single particle interferometric reflectance imaging sensor (SP-IRIS) has been shown to be a sensitive and specific-detection platform in serum and whole blood. This paper presents a step towards a POC viral assay through a SP-IRIS prototype with automated data acquisition and analysis and a simple, easy-to-use software interface. Decreasing operation complexity highlights the potential of SP-IRIS as a sensitive and specific POC diagnostic tool. With the integration of a microfluidic cartridge, this automated instrument will allow an untrained user to run a sample-to-answer viral assay in the POC setting.

Label-free multiplexed virus detection using spectral reflectance imaging.

We demonstrate detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The Interferometric Reflectance Imaging Sensor (IRIS) has been shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format. Vesicular stomatitis virus (VSV) was used as the target for detection as it is well-characterized for protein composition and can be modified to express viral coat proteins from other dangerous, highly pathogenic agents for surrogate detection while remaining a biosafety level 2 agent. We demonstrate specific detection of intact VSV virions achieved with surface-immobilized antibodies acting as capture probes which is confirmed using fluorescence imaging. The limit of detection is confirmed down to 3.5 × 10(5)plaque-forming units/mL (PFUs/mL). To increase specificity in a clinical scenario, both the external glycoprotein and internal viral proteins were simultaneously detected with the same antibody arrays with detergent-disrupted purified VSV and infected cell lysate solutions. Our results show sensitive and specific virus detection with a simple surface chemistry and minimal sample preparation on a quantitative label-free interferometric platform.

Contribution of reactive oxygen species and caspase-3 to apoptosis and attenuated ICAM-1 expression by paclitaxel-treated MDA-MB-435 breast carcinoma cells.

Paclitaxel is a microtubule-stabilizing and apoptosis-inducing drug that is commonly used to treat metastatic breast cancer, although the mechanism of paclitaxel-induced apoptosis remains incompletely understood. Furthermore, adhesion molecule expression is attenuated on mouse mastocytoma and human leukemia cells that survive short-term culture in the presence of paclitaxel. In the present study we show that MDA-MB-435 human breast carcinoma cells that survived culture for 72 h in the presence of submaximal cytotoxic concentrations of paclitaxel (0.02 and 0.01 microg/ml) showed decreased expression of the adhesion molecule ICAM-1. Paclitaxel treatment of MDA-MB-435 cells was associated with the generation of reactive oxygen species (ROS), dissipation of mitochondrial transmembrane potential, and the activation of caspase-3. The antioxidant glutathione protected MDA-MB-435 cells from paclitaxel-induced cytotoxicity and reduced ICAM-1 expression. In addition, a selective inhibitor of caspase-3 (Z-DEVD-FMK), as well as a pan-caspase inhibitor (Z-VAD-FMK), partially prevented the decrease in ICAM-1 expression observed following paclitaxel treatment, but did not protect against paclitaxel-induced cytotoxicity. We conclude that the paclitaxel-induced reduction in ICAM-1 expression by MDA-MB-435 breast carcinoma cells is both ROS- and caspase-dependent, whereas paclitaxel-induced cytotoxicity is ROS-dependent and does not involve caspases. Decreased ICAM-1 expression by breast carcinoma cells that survive paclitaxel treatment may negatively impact on cytotoxic lymphocyte-mediated destruction of paclitaxel-resistant breast cancer cells in the context of chemo-immunotherapy or chemo-adoptive immunotherapy.

Adenosine-induced apoptosis in EL-4 thymoma cells is caspase-independent and mediated through a non-classical adenosine receptor.

Cell death caused by the accumulation of extracellular adenosine is believed to contribute to the profound loss of T lymphocytes in patients with severe combined immunodeficiency disease due to adenosine deaminase deficiency. Although adenosine is known to trigger apoptosis in thymocytes and peripheral T cells, the molecular basis of this effect is not understood. In this study, we show that adenosine-induced apoptosis in mouse EL-4 thymoma cells was associated with the generation of reactive oxygen species and a reduction in mitochondrial transmembrane potential. In addition, cell death was by a caspase-independent mechanism because caspase inhibitors did not protect EL-4 cells from adenosine-induced cytotoxicity. Although reverse transcriptase polymerase chain reaction revealed that EL-4 cells expressed A2b and A3 adenosine receptor subtypes, blockade of A2b and A3 adenosine receptors with receptor-selective antagonists did not attenuate adenosine-induced cell death. Nevertheless, the failure of nucleoside transport inhibitors to prevent adenosine cytotoxicity suggested that adenosine was acting through a cell-surface receptor. In addition, adenosine-induced apoptosis was not due to an accumulation of intracellular cyclic adenosine monophosphate (cAMP) since neither forskolin nor 8-Br-cAMP was cytotoxic for EL-4 cells. Adenosine therefore acts through a non-classical receptor at the cell surface to trigger caspase-independent apoptosis in mouse thymoma cells.

Comment: Impact of the King's fund report on nutrition 2 years after publication.

The King's Fund Report A Positive Approach To Nutrition As Treatment (King's Fund, 1992) generated much interest. However, a recent survey by Hospital Doctor and Nutricia revealed that its impact in hospitals lias been disappointing (Warne, 1994).