Human-Like Receptor Specificity Does Not Affect the Neuraminidase-Inhibitor Susceptibility of H5N1 Influenza Viruses

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Abstract
If highly pathogenic H5N1 influenza viruses acquire affinity for human rather than avian respiratory epithelium, will their susceptibility to neuraminidase (NA) inhibitors (the likely first line of defense against an influenza pandemic) change as well? Adequate pandemic preparedness requires that this question be answered. We generated and tested 31 recombinants of A/Vietnam/1203/04 (H5N1) influenza virus carrying single, double, or triple mutations located within or near the receptor binding site in the hemagglutinin (HA) glycoprotein that alter H5 HA binding affinity or specificity. To gain insight into how combinations of HA and NA mutations can affect the sensitivity of H5N1 virus to NA inhibitors, we also rescued viruses carrying the HA changes together with the H274Y NA substitution, which was reported to confer resistance to the NA inhibitor oseltamivir. Twenty viruses were genetically stable. The triple N158S/Q226L/N248D HA mutation (which eliminates a glycosylation site at position 158) caused a switch from avian to human receptor specificity. In cultures of differentiated human airway epithelial (NHBE) cells, which provide an ex vivo model that recapitulates the receptors in the human respiratory tract, none of the HA-mutant recombinants showed reduced susceptibility to antiviral drugs (oseltamivir or zanamivir). This finding was consistent with the results of NA enzyme inhibition assay, which appears to predict influenza virus susceptibility in vivo. Therefore, acquisition of human-like receptor specificity does not affect susceptibility to NA inhibitors. Sequence analysis of the NA gene alone, rather than analysis of both the NA and HA genes, and phenotypic assays in NHBE cells are likely to adequately identify drug-resistant H5N1 variants isolated from humans during an outbreak. If the avian influenza H5N1 viruses adapt to human hosts, the first step is likely to be a switch in the preference of their viral hemagglutinin (HA) glycoprotein to bind to human rather than avian cell receptors. Such a switch may also alter virus susceptibility to neuraminidase (NA) inhibitors, which are anti-influenza drugs that are likely to be the first line of defense against a pandemic. We generated recombinant A/Vietnam/1203/04-like (H5N1) viruses carrying HA mutations previously shown to alter receptor specificity or affinity. We also discovered a previously unknown route (three simultaneous HA amino acid substitutions) by which highly pathogenic H5N1 viruses can adapt to human receptors. We then used a novel cell-culture–based system (differentiated human airway epithelial NHBE cells) to evaluate the recombinant viruses' resistance to NA inhibitors. None of the HA-mutant recombinants showed reduced drug susceptibility. Our results indicate that the tested HA mutations are unlikely to cause resistance to NA inhibitors in vivo. The NHBE system meets the need for an appropriate cell-culture–based system for phenotypic characterization of drug resistance.