(471g) Single-Particle Assay for Detecting Influenza Virus and Characterizing Its Binding and Fusion Rates

The rapid detection and characterization of enveloped viruses are important for controlling viral pandemics. Here we discuss a single-particle technique for detecting influenza through receptor binding and membrane fusion behaviors. Influenza utilizes surface proteins called hemagglutinin to bind to sialic acid groups on the host cell surface, and to initiate the fusion of the viral and host membrane. Membrane fusion is triggered at a low pH and is required for infection. For the assay, we use a microfluidic device made on a glass substrate that has been coated with a supported lipid bilayer (SLB). The SLB contains sialic acid receptor molecules and can be modified to mimic various host cell membranes. Total internal reflection microscopy is used to monitor the SLB and any fluorescent object that interacts with it. To detect the viral envelope, trace amounts of lipophilic fluorophores were added to the virus-contaminated solution before loading it into the device. We were able to detect individual virions attaching to the SLB, and more importantly, quantify the rate at which binding and unbinding occurs for each virion. To detect membrane fusion activity, an acidic solution was flown over the bound viruses. The spread of the lipophilic fluorophore from the viral membrane to the SLB confirmed fusion activity and the rate at which this occurs was quantified. Using the binding, unbinding, and fusion rate information, one could potentially produce a list of host species that are susceptible to infection and thus implement appropriate quarantine procedures.