Tetrameric neuraminidases cleave the end-capping sialylated monomer from oligosaccharide ligands at the surface of a host cell infected by the influenza A virus. This cleavage releases the replicated virions from the host cell, making drugs that inhibit neuraminidase function effective to treat influenza A infections. A capillary electrophoresis separation-based assay is reported that maintains the native structure of tetrameric viral neuraminidases derived from H1N1 or H5N1 influenza A pandemics which convert, in-real time, a substrate that mimics 6'-sialyllated threonine-linked glycans on human cells. The assay integrates the enzyme reaction with the separation and is operated using a background electrolyte containing 100 mM NaCl with a thermally reversible nanogel in a 10 μm inner diameter fused silica capillary. In addition to defining the 0.4 nL reaction zone maintained at 37 °C, the nanogel medium resolves the substrate from contaminants as well as the substrate from the product before and after the enzymatic conversion. The enzyme activity is quantifiable based on the percent conversion observed in the presence of a range of inhibitor concentrations. For 1918 H1N1 (A/Brevig Mission/1/18) neuraminidase, the inhibition constant of the transition state analog 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) is 3.5 ± 0.8 μM (n = 5). The inhibition constants for oseltamivir acid (inhibiting compound of Tamiflu) and peramivir (Rapivab) are 18.2 ± 0.5 nM (n = 3) and 67 ± 8 nM (n = 3), respectively. For 2004 H5N1 (A/Vietnam/1203/2004) neuraminidase, which contained a foreign tetramerization domain to maintain the structure, the inhibition constant for peramivir is 5.4 nM.

Neuraminidase inhibitors (NAIs) are the main class of antivirals currently used for the treatment of influenza infections. As influenza viruses are constantly evolving, drug-resistance can emerge resulting in reduced effectiveness of treatment. This study evaluated the presence of molecular markers associated with NAI susceptibility in 724 influenza A(H1N1)pdm09 positive samples from Brazilian surveillance system from the 2014-2016 seasons, including 76 isolates tested for oseltamivir (OST) susceptibility and 23 isolates also tested for zanamivir, peramivir and laninamivir susceptibility. We identified the H275Y (n = 3) and I223K (n = 1) NA substitutions, associated with reduced inhibition (RI) by the NAIs. Noteworthy, no epidemiological links were identified among the patients infected with the mutant viruses. Phylogenetic analysis from NA and hemagglutinin genes showed that mutant viruses were not clustered. All mutant virus strains carried the permissive substitutions V241I and N369K, in addition to the N386K, which has been shown to destabilize the NA structure. Functional NA analysis of one virus containing the H275Y mutation confirmed its highly RI profile to OST and peramivir and demonstrated that it had decreased viral replication and NA thermostability compared to the wild type virus. The remaining tested isolates presented normal inhibition profile to the NAIs tested. In conclusion, the overall frequency of influenza A(H1N1)pdm09 viruses bearing mutations associated with NAI RI was 0.6%, similar to what has been observed in recent global studies.
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