An easy, rapid, and sensitive method for detection of drug-resistant influenza virus by using a sialidase fluorescent imaging probe, BTP3-Neu5Ac

Immunochromatographic kits and RT-PCR are widely used as diagnostic tools for influenza detection in clinical and hygiene fields. Immunochromatographic kits are useful for differential typing of influenza A and influenza B but cannot show if the detected virus strains have acquired drug resistance against neuraminidase inhibitors that target sialidase activity of viral neuraminidase. Although RT-PCR enables determination of drug-resistant mutants, its efficacy is limited to viruses carrying a known substitution in their neuraminidase genome sequence. In the present study, an easy, rapid and sensitive method for detection of drug-resistant influenza viruses regardless of major antigenic changes or genomic mutations was developed. By using the method in combination with virus-concentrated membranes in centrifugal filter units and a sialidase imaging probe, 2-(benzothiazol-2-yl)-4-bromophenyl-N-acetylneuraminic acid (BTP3-Neu5Ac), sialidase activity of influenza neuraminidase was visualized on membranes by the green fluorescence of produced hydrophobic BTP3 under UV irradiation with a handheld UV flashlight. Fluorescence images in the presence or absence of neuraminidase inhibitors clearly discriminated drug-resistant influenza viruses from drug-sensitive ones. The assay can be done within 15 min. The detection sensitivity was shown to be equal to or higher than the sensitivities of commercial immunochromatographic kits. The assay will be a powerful tool for screening and monitoring of emerging drug-resistant influenza viruses and would help clinicians decide effective antiviral treatment strategies when such mutants have become prevalent.