A Rapid and Sensitive Flu Virus Test

NON-TECHNICAL SUMMARY: Avian influenza is a highly contagious disease that can spread rapidly in a susceptible population. Thus, avian influenza may have devastating effects on the poultry industry. Because of potential interspecies transmission, as evidenced with the avian influenza virus H5N1, avian influenza may have devastating effect on public health as well. It is therefore important to prevent and/or to promptly contain avian influenza. Prompt and accurate identification of an infected flock is crucial for eradicating avian influenza, which depends on the availability of rapid and reliable diagnostic tests. Ideally, an effective avian influenza test should be very sensitive, rapid (e.g., <30 minutes), economical, portable, compatible with tracheal or cloacal swab samples, and easy-to-use. Currently available tests do not meet these criteria. Current tests are mostly immunoassays and molecular assays, which suffer from a number of drawbacks such as lack of sensitivity (e.g., lateral flow based immunoassays), use of expensive equipment, and susceptibility to inhibitors found in the samples (e.g., molecular assays). We have developed a prototype test kit. Preliminary evaluation of the kit using human influenza virus has demonstrated that the assay is highly sensitive, rapid, portable, and easy-to-use. The proposed Phase I studies will evaluate the kit!=s performance using chicken tracheal swabs spiked with avian influenza virus and other samples containing avian influenza virus. Successful completion of the Phase I studies will lead to Phase II studies, which will evaluate the test kit using samples from chickens infected with avian influenza virus. Successful development of the test kit will provide the poultry industry with essential ammunition against avian influenza. <P>

APPROACH: The substrates will be synthesized and purify by our chemist using protocol described in the preliminary study section of the proposal; we have used the protocol to successfully synthesize the substrate. The test kit will then be evaluated by determining its analytical sensitivity and performance on contrived samples. An isolate of avian flu strain H6N2 at 0, 10, 25, 50, 100, 250, 500, 1000, 5000, or 50,000 embryo lethal dose (ELD50)/mL will be used for the analytical sensitivity study. 200 microliters of sample will be added to a detection vial containing lyophilized detection mix. After incubation at room temperature for 5 minutes, the detection vial will be placed in luminometer for measurement of light signal. Twenty four (24) replicates will be tested for each concentration. Mean RLU (the light signal) and standard deviation (SD) for the 24 negative samples will be determined using the MS Excel program. Cutoff values will be set at 2.6 or 1.96 standard deviations above the mean (Fig. 4), which will give a specificity of 99% and 95% respectively. The study with the contrived samples will be carried out as follows: Twenty-four (24) tracheal swab samples will be collected from 8 healthy chickens (3 samples from each chicken). 50 µL of PBS buffer containing 0, 50, 100, 500, 1000, 5000, 10,000, or 20,000 ELD50 units of avian flu virus H6N2 will be spiked into each swab. Triplicates of spiked samples will be prepared and tested for each viral concentration.