Methods for determining resistance to neuraminidase inhibitors

Abstract

Various phenotypic methods have been used to monitor viral susceptibility to anti-viral agents in the management of virus diseases. Confirmation of resistance generally requires genotyping of the inhibitor target gene(s) and identification of mutations that confer the phenotypic resistance. While plaque reduction assays are valuable for accurate measurements of susceptibility, they are not ideal for large-scale monitoring. For the influenza inhibitors amantadine/rimantadine, susceptibility of clinical isolates has been monitored predominantly by yield-reduction using EIA-based assays, because plaque formation by influenza clinical isolates is very variable. With the development of the influenza neuraminidase (NA) inhibitors (NIs), it became apparent that current cell-based assays were unsuitable for monitoring susceptibility to this new class of drugs. The reasons for this relate to the close functional interactions between the NA and the HA at virus release and entry, and that weak HA receptor binding may by-pass the NA function in cell assays. Mutations selected in the HA, while not apparently contributing to phenotypic resistance in vivo, may result in cell culture-based resistance, and conversely may mask NA resistance in cell culture by modifying receptor-binding specificity. One important distinction between NIs and other antiviral enzyme inhibitors is that both target enzyme and inhibitor work extracellularly. Therefore, direct NA enzyme susceptibility assays using whole virus are most representative of the in vivo situation for monitoring phenotypic susceptibility. Sequencing of the NA to confirm phenotypic resistance is important. Since no suitable phenotypic assay is available to measure changes in receptor binding, sequencing of the HA, in particular the receptor binding region is an important adjunct to help understand resistance mechanisms. As the clinical use of NIs escalates, a major change will be required in approaches used to monitor susceptibility of influenza isolates in virology laboratories worldwide.