Neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir

Abstract

The influenza virus neuraminidase (NA) inhibitors zanamivir and oseltamivir were introduced into clinical practice in various parts of the world between 1999 and 2002. In order to monitor the potential development of resistance, the Neuraminidase Inhibitor Susceptibility Network was established to coordinate testing of clinical isolates collected through the World Health Organization influenza surveillance network from different regions of the world (M. Zambon and F. G. Hayden, Antivir. Res. 49:147-156, 2001). The present study establishes the baseline susceptibilities prior to and shortly after the introduction of the NA inhibitors. Over 1000 clinical influenza isolates recovered from 1996 to 1999 were tested. Susceptibilities were determined by enzyme inhibition assays with chemiluminescent or fluorescent substrates with known NA inhibitor-resistant viruses as controls. The 50% inhibitory concentrations (IC(50)s) depended upon the assay method, the drug tested, and the influenza virus subtype. By both assays, the mean zanamivir IC(50)s were 0.76, 1.82, and 2.28 nM for the subtype H1N1 (N1), H3N2 (N2), and B NAs, respectively, and the oseltamivir IC(50)s were 1.2, 0.5, and 8.8 nM for the N1, N2, and B NAs, respectively. The drug susceptibilities of known zanamivir- and oseltamivir-resistant viruses with the NA mutations E119V, R292K, H274Y, and R152K fell well outside the 95% confidence limits of the IC(50)s for all natural isolates. Sequence analysis of the NAs of viruses for which the IC(50)s were above the 95% confidence limits and several control isolates for which the IC(50)s were in the normal range revealed variations in some previously conserved residues, including D151, A203, T225, and E375 (N2 numbering). Known resistance mutations are both influenza virus subtype and drug specific, but there was no evidence of naturally occurring resistance to either drug in any of the isolates.

FIG. 1.

Box plots of the log₁₀ of the mean IC₅₀s of each of the inhibitors for each virus type by the fluorescent and chemiluminescent assays. The ends of the solid lines extending on either side of the box represent the approximate 95% confidence limits. Extreme values are those that lie outside the 95% confidence limits. The position of the text label for each mutant corresponds to the IC₅₀. w1 and w2, values for paired wild-type controls for mutants.

FIG. 2.

Grid plots showing the correspondence between inhibition by the chemiluminescent and fluorescent assays for individual samples. Each sample was assayed in duplicate. The four vertical and horizontal lines refer to the upper and lower 95% confidence limits and the 25th and 75th percentiles. Values lying outside the upper and lower 95% confidence limits and the four corners of the plots show where an extreme value by one assay corresponds to an extreme value by the other assay for a particular isolate. The IC₅₀s of at least one of the drugs fall in the upper extreme values for mutant NAs. The position of the text label for each mutant corresponds to its IC₅₀s by both assays. w1 and w2, values for paired wild-type controls for mutants.

FIG. 3.

X-ray crystallographic model of influenza virus B/Beijing/87 NA (Protein Data Bank reference 1NSC). The amino acids at positions 203 and 375, which have coevolved, as well as residues D151 and T225 (positions 201, 377, 148, and 223, respectively, in the numbering for influenza B virus), were mapped. Since positions 203 and 375 fall on opposite sides of the head, they are clearly not compensatory variations.