Abstract
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.
Highlights
Influenza virus is a major cause of severe and acute respiratory tract infections
Complications of influenza virus infection can result in viral pneumonia, secondary bacterial pneumonia, encephalopathy in infants, or sinusitis, which may lead to increased mortality, especially among people in high-risk groups [1,2,3,4]
In order to examine the optimal concentration of calcium ion and the optimal temperature for BTP3-Neu5Ac reaction, we incubated influenza A virus (IAV) or influenza B virus (IBV) with 100 μM BTP3-Neu5Ac under varying conditions of CaCl2 concentration and temperature
Summary
Influenza virus is a major cause of severe and acute respiratory tract infections. Complications of influenza virus infection can result in viral pneumonia, secondary bacterial pneumonia, encephalopathy in infants, or sinusitis, which may lead to increased mortality, especially among people in high-risk groups [1,2,3,4]. Since the first reported case of human infection with avian IAV (H7N9) in 2013, the number of patients with H7N9 infection has been increasing, especially in China [7]. This suggests the pandemic potential of new subtypes of human IAV that originated in avian IAV. Epidemic influenza infection, which is highly contagious, show rapid progression of symptoms, and has the potential to induce encephalopathy, especially in infants, should be diagnosed at an early stage of disease onset and appropriate medication with anti-influenza agents should be initiated as soon as possible [4,9]
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