Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses

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.

Figure 1

(A) Crystal structure of the A/Vietnam/1203/04 hemagglutinin molecule (Protein Data Bank:2FK0) showing 11 positions in or near the receptor binding site (yellow) at which amino acid substitutions can alter receptor specificity/affinity. Residues in red are positions where a single substitution affects the stability of the H5N1 virus. (B) Affinity of recombinant A/Vietnam/1203/04-like (H5N1) influenza A viruses for high molecular weight sialic acid substrates, both natural (fetuin) and synthetic. Of the nine substrates tested (Table S1), only the five shown here had a K_ass significantly different from that of the negative control. Bars represent the association constants (K_ass) of virus in complex with sialylglycopolymer. Values are the means±s.d. of four independent experiments.

Figure 2. Susceptibility of recombinant A/Vietnam/1203/04-like (H5N1) influenza A viruses to NA inhibitors in (A) MDCK cells and (B) MDCK-SIAT1 cells as determined by plaque reduction assay.

EC₅₀ was the dose of drug required to reduce plaque size by 50%. Values are the means±s.d. of three independent experiments. * P<0.01 compared to wild-type rgVN1203 virus (one-way ANOVA performed for all viruses); °P<0.01 compared to virus carrying only the NA H274Y substitution (one-way ANOVA performed for viruses carrying the H274Y NA mutation).

Figure 3. Susceptibility of recombinant A/Vietnam/1203/04-like (H5N1) influenza A viruses to NA inhibitors in (A) A549 and (B) NHBE cells as determined by virus reduction assay.

In A549 cells, EC₅₀ was the dose of drug that reduced the TCID₅₀ titer of culture supernatant 50% as compared to no-drug controls 72 h after infection. In NHBE cells, virus yield was measured after 24 h incubation with 0, 0.1, 1, or 10 µM of NA inhibitor by TCID₅₀ assay in MDCK cells. All concentrations yielded similar results; those obtained with 1 µM NA inhibitor are shown. * P<0.01 compared to wild-type rgVN1203 virus, one-way ANOVA test performed for all viruses; °P<0.01 compared to H274Y virus, one-way ANOVA test performed for viruses carrying the H274Y NA mutation.