Natural variation can significantly alter the sensitivity of influenza A (H5N1) viruses to oseltamivir

Abstract

Geographic spread of highly pathogenic avian H5N1 influenza viruses may give rise to an influenza pandemic. During the first months of a pandemic, control measures would rely mainly on antiviral drugs, such as the neuraminidase (NA) inhibitors oseltamivir and zanamivir. In this study, we compare the sensitivities to oseltamivir of the NAs of several highly pathogenic H5N1 viruses isolated in Asia from 1997 to 2005. The corresponding 50% inhibitory concentrations were determined using a standard in vitro NA inhibition assay. The K(m) for the substrate and the affinity for the inhibitor (K(i)) of NA were determined for a 1997 and a 2005 virus, using an NA inhibition assay on cells transiently expressing the viral enzyme. Our data show that the sensitivities of the NAs of H5N1 viruses isolated in 2004 and 2005 to oseltamivir are about 10-fold higher than those of earlier H5N1 viruses or currently circulating H1N1 viruses. Three-dimensional modeling of the N1 protein predicted that Glu248Gly and Tyr252His changes could account for increased sensitivity. Our data indicate that genetic variation in the absence of any drug-selective pressure may result in significant variations in sensitivity to anti-NA drugs. Although the clinical relevance of a 10-fold increase in the sensitivity of NA to oseltamivir needs to be investigated further, the possibility that sensitivity to anti-NA drugs could increase (or possibly decrease) significantly, even in the absence of treatment, underscores the need for continuous evaluation of the impact of genetic drift on this parameter, especially for influenza viruses with pandemic potential.

FIG. 1.

Phylogenetic relationships among N1 genes of influenza A viruses (nucleotides 6 to 1334 from ATG). Published sequences were retrieved from the Los Alamos influenza virus sequence database (http:/www.flu.lanl.gov). The dendrogram was constructed by the genetic distance matrix method, calculated with the DNADIST program, using the Kimura two-parameter model with a transition-to-transversion ratio of 2.0, and by neighbor-joining analysis in the PHYLIP package (3). The unrooted tree has the N1 protein from A/NewCaledonia/20/99 as an outgroup. Bootstrap values for 100 replicates are given at the nodes. The IC₅₀ for OC measured for each virus is indicated as the mean ± standard deviation (SD) for two (*) or three independent determinations. For each of the three indicated groups of viruses (H1N1 viruses, 1997-2003 H5N1 viruses, and 2004-2005 H5N1 viruses), the average IC₅₀ for OC (mean ± SD) is shown. Viruses for which the K_m for substrate and K_i for OC were determined are underlined.

FIG. 2.

Overlay of predicted three-dimensional structures of the N1 proteins of HK156/97 and C408/05 in complex with oseltamivir carboxylate. (A) Global view of N1 structure, as predicted using the SWISS Model server and the crystallographic coordinates of A/Tern/Australia/G70C/75 N9 in complex with OC (in gray) as a template. The membrane plan is normal to the indicated fourfold axis, on the opposite side of the protein. OC is located at the active site of the N1 protein. Amino acid differences between the N1 proteins of HK156/97 and C408/05 predicted to have a significant impact on binding to OC are localized within the subdomain represented in blue (β₃S₂ and β₃S₃ β-sheets linked by the β₃L₂₃ loop). (B) Enlarged representation of the boxed region in panel A. Amino acids from HK156/97 are represented in red (Glu248 and Tyr252), whereas the residues found at the same positions in C408/05 are represented in yellow (Gly248 and His252). The hydrogen bond between residue Ser246 of the active site and the O-ethyl-propyl group of OC is represented by a dotted line. This interaction is favored in the C408/05-derived N1 model, with residue Gly248 conferring flexibility to the β₃L₂₃ loop and residue His252 interacting with Thr242, thus strengthening the underlying structure formed by the antiparallel β₃S₂ and β₃S₃ β-sheets. In contrast, in the HK156/97-derived N1 model, residue 252 is a Tyr involved in a stacking interaction with residue Tyr275 opposite Ser246, pulling Ser246 away from OC. Moreover, residue Glu248, replacing Gly248, likely renders the loop around Ser246 more rigid than in the C408/05-derived N1 protein.