Monitoring of viral susceptibility: new challenges with the development of influenza NA inhibitors.

With the clinical development of anti-viral agents, monitoring for the continued susceptibility of wild-type strains has become important in disease management. Various methods have been used to monitor viral susceptibility; the advantages and disadvantages of which depend on the virus, the target and the scale of the research being undertaken. The plaque-reduction assay is valuable for measuring susceptibility of most viruses but is not ideal for large-scale monitoring. Yield-reduction, measuring specific virus antigens, and dye-uptake assays, measuring virus cytopathic effects, are more suitable for high-throughput requirements, but the IC(50) value (the concentration that inhibits 50% of virus) varies with the viral inoculum. Surveillance of influenza susceptibility to rimantadine/amantadine in the clinic has predominantly used EIA-based assays, since plaquing of influenza clinical isolates is variable. With development of the influenza NA inhibitors it became apparent that current cell-based assays were unsuitable for monitoring susceptibility to this new class of drugs. Variability may result from virus spread directly from cell to cell in culture by-passing the NA function. Furthermore, mutations selected in the HA, while not apparently contributing to phenotypic resistance in vivo, may result in cell-culture based resistance, and may mask NA resistance in cell culture by modifying receptor-binding specificity. One important distinction between NA inhibitors and other antiviral enzyme inhibitors is that both target enzyme and inhibitor work extracellularly. NA assays are therefore most representative of the in vivo situation for monitoring susceptibility, supported by HA sequencing. As the clinical use of NA inhibitors escalates, a major change will be required in approaches used to monitor susceptibility of influenza isolates in virology laboratories world-wide.