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Antimicrobial Agents and Chemotherapy, November 2006, p. 3809-3815, Vol. 50, No. 11
0066-4804/06/$08.00+0     doi:10.1128/AAC.00645-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Natural Variation Can Significantly Alter the Sensitivity of Influenza A (H5N1) Viruses to Oseltamivir{triangledown}

M. A. Rameix-Welti,1 F. Agou,2 P. Buchy,3 S. Mardy,3 J. T. Aubin,1 M. Véron,2 S. van der Werf,1,4 and N. Naffakh1*

Unité de Génétique Moléculaire des Virus Respiratoires, URA CNRS 1966,1 Unité de Régulation Enzymatique des Activités Cellulaires, URA CNRS 2185, Institut Pasteur, Paris, France,2 Institut Pasteur du Cambodge, Phnom Penh, Cambodia,3 EA302, Université Paris 7, Paris, France4

Received 26 May 2006/ Returned for modification 7 July 2006/ Accepted 17 August 2006


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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 Km for the substrate and the affinity for the inhibitor (Ki) 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.


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INTRODUCTION
 
Since 1997, highly pathogenic avian influenza A viruses of the H5N1 subtype have been spreading from Southeast Asia to Europe and, most recently, the Middle East and Africa, causing large outbreaks among domestic poultry and sporadic transmission from poultry to humans (1). Cases of human infections caused by an avian H5N1 virus were recorded in Hong Kong in December 1997 (18 cases, with 6 fatalities) and later in February 2003 (2 cases, with 1 fatality). Since the end of 2003, a total of 247 laboratory-confirmed infections, 144 of which were fatal, were reported to the World Health Organization by public health authorities in Vietnam, Thailand, Cambodia, Indonesia, China, Azerbaijan, Turkey, Iraq, Egypt, and Djibouti (32a). Human-to-human transmission of the virus has been very inefficient so far. However, genetic mutations or reassortments could lead to the emergence of H5N1 viruses with increased transmissibility among humans and with the potential to initiate a global influenza pandemic.

The options for efficient control of such an emerging influenza threat include vaccination and antiviral treatment. Reverse genetics has been used to produce H5N1 candidate vaccine strains which proved efficient in animal models (10, 17) and are currently undergoing clinical trials. Such candidate vaccines were shown to elicit protective antibody levels in humans, albeit only after multiple injections with high doses of antigen and/or in the presence of adjuvants (25). However, because H5N1 viruses undergo continuous antigenic drift, the production of a matched vaccine strain will be required at the onset of the pandemic, and the vaccine would therefore not be available until a few months later. Thus, during this early period, specific control measures would rely solely on antiviral drugs. Zanamivir and oseltamivir are sialic acid analogues that selectively target the neuraminidase (NA) enzyme (16, 27). Both have been shown to be safe and effective drugs for the prophylaxis and early treatment of H3N2 and H1N1 influenza virus infections in humans (20). Animal studies suggest that neuraminidase inhibitors could also be effective against avian H5N1 viruses (6, 8, 14, 33). Therefore, stockpiling of antineuraminidase drugs is a key element in the recently revised pandemic preparedness plans of several countries (30).

The effectiveness of a containment strategy based on massive therapeutic and prophylactic use of antiviral drugs will depend on the accuracy of the dosage, and thus on the precise evaluation of the actual sensitivities of the targeted viruses to the drugs. The sensitivity of the neuraminidases of potentially pandemic H5N1 influenza viruses isolated so far to oseltamivir has already been established using in vitro inhibition assays (6, 8, 14). However, the question of how natural variation in N1 could affect its sensitivity has not been addressed thoroughly. In the present study, we compare the sensitivities to neuraminidase inhibitors of several highly pathogenic H5N1 viruses isolated in Asia throughout the 1997-2005 period. Our results show significantly increased sensitivities of the neuraminidases of the more recent H5N1 viruses to oseltamivir carboxylate (OC; the active metabolite of oseltamivir) that could be related to specific changes in their N1 antigens.


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MATERIALS AND METHODS
 
Viruses. Pre-2004 H5N1 influenza viruses A/HongKong/156/97 and A/HongKong/213/03 were kindly provided by Alan Hay (NIMR, London, United Kingdom) and Wilina Lim (Hong Kong National Influenza Center, Hong Kong), respectively. Post-2004 H5N1 viruses A/VietNam/JP14/05, A/VietNam/JP20-2/05, A/VietNam/JP4207/05, and A/VietNam/JPHN/30321/05 were kindly provided by Masato Tashiro (WHO Collaborative Center for Reference and Research on Influenza, National Institute of Infectious Diseases, Tokyo, Japan); the A/VietNam/1203/04 virus was kindly provided by Wilina Lim; and viruses A/Ck/Cambodia/07/04, A/Goose/Cambodia/26/04, A/Ck/Cambodia/013LC1b/05, A/Ck/Cambodia/013LC2b/05, and A/Cambodia/408008/05 were isolated from specimens provided by the Institut Pasteur in Cambodia. The H1N1 influenza virus used as a reference (A/Paris/0650/04) was isolated by the National Influenza Center (Northern France) at the Institut Pasteur in Paris (France). Viruses were propagated in MDCK cells. Experiments with infectious H5N1 influenza viruses were conducted in a biosafety level 3+ facility approved for studies of these viruses.

Neuraminidase in vitro inhibition assay. NA enzymatic activity was measured using the fluorogenic substrate 2'-(4-methylumbelliferyl)-{alpha}-D-N-acetylneuraminic acid (MUNANA; Sigma), as previously described (21). The fluorescence of the released 4-methylumbelliferone was measured using a Xenius spectrofluorometer (SAFAS) at excitation and emission wavelengths of 330 and 450 nm, respectively. For NA inhibition assays, viral suspensions were adjusted to equivalent NA contents in MES buffer (33 mM morpholineethanesulfonic acid, pH 6.5, 120 mM NaCl, 4 mM CaCl2), based on preliminary determinations of the NA activities in serial dilutions of the viral stocks. Viral suspensions were preincubated in the presence of various concentrations of OC (0.01 to 100 nM; Hoffman-La Roche) for 1 h at 37°C in 96-well plates. Following the addition of substrate at a final concentration of 100 µM, viruses were incubated for 1 h at 37°C, and the reaction was stopped by adding 1 volume of a solution of 1 M glycine, pH 10.7, and 25% ethanol. In order to inactivate the viruses, NP-40 was added to a final concentration of 0.5%, and samples were incubated for 1 h at room temperature and then for 1.5 h at 62.5°C. Fluorescence values were measured, and the 50% inhibitory concentration (IC50) for NA enzymatic activity was determined from the dose-response curve, using KaleidaGraph software (Synergy Software).

Cell-based virus inhibition assay. MDCK cells were infected with 0.001 PFU/cell of virus at 35°C. At 1 hour postinfection, they were incubated in the presence of serial dilutions of oseltamivir carboxylate (10 µM to 0.1 nM) for 72 h at 35°C. Virus replication was evaluated by measuring the hemagglutination activity in the supernatants. The 50% effective concentration (EC50) of oseltamivir was determined as the lowest concentration of oseltamivir carboxylate which induced a significant shift of the hemagglutination titer (>4-fold).

Neuraminidase enzymatic activity and inhibition assays using whole cells transiently expressing the viral enzyme. The sequence encoding neuraminidase was amplified from viral RNA by reverse transcription-PCR and cloned into the pCI vector (Invitrogen), using standard procedures (22). Two independent clones were selected for the N1 protein of each of the three viruses included in the study. The His274Tyr mutation was introduced into each clone by site-directed mutagenesis, using a QuickChange site-directed mutagenesis kit (Stratagene). All constructs were verified by sequencing using a Big Dye Terminator sequencing kit and an automated sequencer (Perkin-Elmer).

Subconfluent monolayers of 293T cells were transfected with pCI-N1 plasmids by using the FuGENE 6 transfection reagent (Roche). At 24 h posttransfection, cells were harvested, and a fraction was used to analyze NA surface expression by an indirect immunofluorescence assay, using a rabbit polyclonal serum directed against the A/New Caledonia/20/99 (H1N1) virus and a FACSCalibur fluorocytometer (Becton Dickinson). Half of the remaining cells were resuspended in MES-DM buffer (MES buffer containing 0.92 g/liter ß-dodecyl-D-maltoside) to prepare soluble NA-containing extracts (MES-DM extracts). Samples were incubated for 1 h at 4°C with gentle shaking and cleared by centrifugation at 13,000 x g for 10 min.

NA enzymatic activity was measured in cell suspensions or MES-DM cell extracts, using the MUNANA fluorogenic substrate as described above. The final concentration of the substrate ranged from 5 to 100 µM. Fluorescence was monitored every 45 s for 20 to 30 min at 37°C, using a Xenius spectrofluorometer (SAFAS) with excitation and emission wavelengths of 330 and 450 nm, respectively. To measure the inhibitory effect of OC or zanamivir (GlaxoSmithKline), cells were preincubated for 30 min at 37°C in the presence of various concentrations of the drugs (0.1 to 10 nM). The kinetic parameters Vmax, Km, and Ki were calculated by fitting the data to the appropriate Michaelis-Menten equations, using the Levenberg-Marquardt algorithm as provided in the commercially available KaleidaGraph software package (Synergy Software).


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RESULTS AND DISCUSSION
 
The sensitivities to oseltamivir of the neuraminidases of human and related avian influenza A (H5N1) viruses isolated in Asia throughout the 1997-2005 period were evaluated using an in vitro NA inhibition assay. The following viruses were included in the study: two pre-2004 human isolates, A/Hong Kong/156/97 and A/Hong Kong/213/03; six post-2004 human isolates, A/Vietnam/1203/04, A/VietNam/JP14/05, A/VietNam/JPHN/30321/05, A/VietNam/JP4207/05, A/VietNam/JP20-2/05, and A/Cambodia/408008/05; and four closely related post-2004 avian isolates, A/Ck/Cambodia/07/04, A/Goose/Cambodia/26/04, A/Ck/Cambodia/013LC1b/05, and A/Ck/Cambodia/013LC2b/05. Phylogenetic relationships among the corresponding NA genes are shown in Fig. 1. Viruses isolated in 2004 and 2005 all belong to genotype Z (15) and genetic clade 1, as defined by H5 phylogeny (32). The NA coding sequences from the most distantly related viruses (A/Vietnam/JP14/05 and A/Goose/Cambodia/26/04) differ by 18 nucleotides (2% divergence) and 8 amino acids (2% divergence). The A/Hong Kong/213/03 and A/Hong Kong/156/97 viruses belong to distinct genotypes (Z+ and Gs/Gd, respectively) and clades (1' and 3, respectively) (15, 32). The corresponding NA coding sequences show 6 and 13% nucleotide divergence (5 and 10% amino acid divergence), respectively, from the NA sequence of A/Vietnam/JP14/05.


Figure 1
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  		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 Km for substrate and Ki for OC were determined are underlined.

In addition to the H5N1 viruses mentioned above, a common influenza A (H1N1) virus isolated in 2004 (A/Paris/0650/04) was included in NA inhibition assays as a standard. The results are presented in Fig. 1. The concentrations of OC required to inhibit the NA enzymatic activities of the A/Paris/0650/04, A/Hong Kong/156/97, and A/Hong Kong/213/03 viruses were similar, with mean IC50 values in the 1.3 to 2.3 nM range, in agreement with previously published data (4, 7, 18, 31). In contrast, the mean IC50 values for H5N1 viruses isolated in 2004 and 2005 ranged from 0.09 to 0.14 nM. Statistical analysis using Student's t test indicated that the sensitivity to oseltamivir of each of the NAs of H5N1 viruses isolated in 2004-2005 was significantly increased compared to those of the NAs of the H5N1 viruses isolated in 1997 and 2003 or of currently circulating H1N1 viruses (P < 0.01).

In order to determine the enzymatic parameters for the NAs of H5N1 viruses and to characterize more precisely the difference in sensitivity to oseltamivir between pre-2004 and post-2004 viruses, we set up an NA inhibition assay with cells transiently expressing the viral enzyme, similar to the assay described by Wang et al. (29). Expression plasmids for the NA proteins derived from the A/Hong Kong/156/97 (HK156/97) and A/Cambodia/408008/05 (C408/05) viruses, which both harbor a deletion in their stalk and are sensitive and highly sensitive to oseltamivir, respectively, as well as from the H1N1 A/Paris/0650/04 virus (P650/04) were transfected into 293T cells. Kinetic analysis of sialidase activity using the MUNANA fluorogenic substrate in the absence or presence of neuraminidase inhibitors was performed on cell suspensions or on solubilized cell extracts as described in Materials and Methods. For the three viral NA proteins, we determined the Michaelis-Menten constant (Km), which reflects the affinity for the substrate, the inhibition constants (Ki) for OC and zanamivir, and the enzymatic activity (Vmax). Our assay was validated by the finding of very similar Km and Ki values for the neuraminidase of P650/04 when enzymatic activity was measured with the whole virus, with whole transfected cells, or with transfected cell extracts. Moreover, our values were close to the Km values for the MUNANA substrate (25 to 30 µM) and Ki values for OC (0.3 to 0.5 nM) reported earlier for neuraminidases of human H1N1 viruses (19, 29), although Ives et al. reported a Km of ~100 µM for the MUNANA substrate (11). This system thus allows enzymatic and genetic studies of the neuraminidase in a native conformation.

The Km and Ki parameters determined with solubilized cell extracts for the neuraminidases of the P650/04, HK156/97, and C408/05 viruses are shown in Table 1. Wild-type neuraminidases showed similar affinities for the substrate (with Km values in the 15 to 21 µM range) and for zanamivir (with Ki values in the 0.2 to 0.35 nM range), whereas they differed markedly in their affinities for OC. The Ki for OC measured for C408/05 (0.073 ± 0.002 nM) was significantly lower than those measured for HK156/97 and P650/04 (0.37 ± 0.05 and 0.31 ± 0.02 nM, respectively; P < 0.001). This finding was in agreement with our previous observations on the IC50 (Fig. 1) and confirmed that the NA of C408/05 is more sensitive to the drug.


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  		TABLE 1. Enzymatic parameters measured for wild-type and His274Tyr mutant N1 recombinant neuraminidases

The NAs of C408/05 and HK156/97 have 12 amino acid differences in their globular domains. In an attempt to predict which amino acid changes could account for the difference in sensitivity to oseltamivir between the two enzymes, we worked out a three-dimensional model of the C408/05 and HK156/97 NAs in complex with OC, using the SWISS Model server with Gromos96 energy minimization (23) and the crystallographic coordinates of the A/Tern/Australia/G70C/75 N9 neuraminidase in complex with OC as a template (PDB no. 2QWK) (26). Amino acids in close contact (<6 Å) with the inhibitor were conserved between C408/05 and HK156/97. Among the amino acids surrounding the active site, residues 248 and 252 (Glu and Tyr, respectively, in HK156/97 and Gly and His, respectively, in C408/05) were predicted to have a significant impact on the molecular interactions with the inhibitor in our model (Fig. 2). We hypothesize that the interaction of Ser246 with the O-ethyl-propyl group of OC contributes to the affinity of NA for the inhibitor and that the nature of residues 252 and 248 determines the strength of this interaction, thus explaining the ~15-fold difference in sensitivities of the NAs of C408/05 and HK156/97 to the inhibitor. Interestingly, an H5N1 isolate which is very close genetically to C408/05, but with a Ser246Gly substitution in the NA, showed an eightfold higher IC50 of OC than that for C408/05 (data not shown), consistent with a role of Ser246 in binding of the inhibitor. Our model could also accommodate the fact that the Ki values for zanamivir, unlike those for OC, are identical for the neuraminidases of the HK156/97 and C408/05 viruses. Indeed, the O-ethyl-propyl group at the 6' position of a cyclohexene ring in OC is replaced with a hydrophilic glycerol side chain at the 6' position of a sugar ring in zanamivir (27). This difference, together with the replacement of the 4' amino group in OC by a 4' guanidino group in zanamivir, is likely to generate different hydrogen bond networks around the inhibitors and to explain the fact that substitutions at residues 252 and 248 of the NA affect the sensitivity to OC but not to zanamivir.


Figure 2
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  		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 (ß3S2 and ß3S3 ß-sheets linked by the ß3L23 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 ß3L23 loop and residue His252 interacting with Thr242, thus strengthening the underlying structure formed by the antiparallel ß3S2 and ß3S3 ß-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.

The isolation of drug-resistant viruses with the His274Tyr substitution was described recently for patients infected with influenza A (H5N1) virus who were treated with oseltamivir (2, 13). The IC50 values for H5N1 viral clones with Tyr at position 274 in their neuraminidases were reported to be above 700 nM, compared to 0.6 nM for His274-containing clones (13). We examined the effect of the His274Tyr mutation on the enzymatic parameters of the N1 protein included in our study and asked whether the difference in sensitivity to oseltamivir between pre-2004 and post-2004 viruses would persist in the presence of the mutation. To this end, expression plasmids for His274Tyr mutant NA proteins derived from the HK156/97, C408/05, and P650/04 viruses were transfected into 293T cells along with expression plasmids for the corresponding wild-type NA proteins for the NA inhibition assay described above. The His274Tyr mutation induced a 300- to 500-fold increase in the Ki values for OC (Table 1) and suppression of the slow-binding phase characteristic of the wild-type enzyme (data not shown) but did not considerably alter the affinity for zanamivir of any of the three NAs tested, in agreement with previously published data (11, 29). The Ki of OC for the mutated C408/05 NA was 24 ± 1 nM, which is significantly lower than the Ki values for the mutated NAs of HK156/97 (127 ± 13 nM) and P650/04 (148 ± 10 nM) (P < 0.001) (Table 1). Thus, the C408/05 NA remained more sensitive to oseltamivir than its HK156/97 and P650/04 counterparts in the presence of the His274Tyr mutation, although all three mutant enzymes were clearly resistant to oseltamivir compared to the wild type.

Resistant H1N1 variants have been shown to be less infectious in cell culture and in animals than sensitive parental viruses (9). This could be related to a negative effect of the His274Tyr mutation on the Km for the substrate and/or the activity (Vmax) of the neuraminidase. In our assay, the His274Tyr mutation resulted in a twofold reduction of the affinity for the substrate of the P650/04 NA (the Km was 40 ± 1.6 for the mutated NA and 21 ± 0.4 µM for the wild type), as already shown by Wang et al. for the neuraminidase of the H1N1 WSN virus (29). A similar level of reduction was observed for HK156/97 NA (Table 1). Remarkably, however, the His274Tyr mutation had no effect on the affinity for the substrate of C408/05 NA, with the mutated and wild-type forms of the enzyme both showing Km values close to 15 µM (Table 1). The effects of the His274Tyr mutation on maximal velocity (Vmax), which is determined by both the specific activity and the amount of enzyme in the reaction, could also be compared. As monitored systematically at the surfaces of transfected cells by fluorescence-activated cell sorting analysis and confirmed by Western blotting analysis of MES-DM cell extracts, the levels of expression of the wild type and the His274Tyr variant of a given NA were similar (data not shown). The mutant-to-wild-type Vmax ratio was 0.5 for C408/05 NA, compared to 0.7 for HK156/97 and P650/04 NA (P < 0.01), suggesting that the activity of the C408/05 NA was more impaired by the His274Tyr mutation. Our findings suggest that for H5N1 viruses, as for H1N1 viruses, the development of resistance to oseltamivir is costly with respect to natural enzymatic activity, and hence is expected to be costly in terms of viral fitness. However, the mechanisms and degree of NA impairment resulting from the His274Tyr mutation may vary depending on sequence variations in the NA.

In the prospect of an influenza pandemic, the accuracy of the dosage of antiviral drugs should be evaluated carefully. Human and related avian H5N1 viruses isolated in Asia throughout the 1997-2005 period were tested for oseltamivir IC50. In addition, the Km for the substrate and the affinity for the inhibitor (Ki) of the NA were determined for a 1997 and a 2005 virus. To our knowledge, these enzymatic constants have not been described for H5N1 viruses so far. We found that the sensitivity to oseltamivir of the NAs of H5N1 influenza A viruses isolated in 2004 and 2005 is about 10-fold higher than that of NAs of earlier H5N1 viruses. Using a subset of two pre-2004 (A/HongKong/156/97 and A/HongKong/213/03) and three post-2004 (A/Vietnam/1203/04, A/Cambodia/408008/05, and A/VietNam/JPHN/30321/05) viruses, we determined the EC50 of oseltamivir on viral replication on cultured MDCK cells, based on the measurement of hemagglutination activity 72 h after infection with 0.001 PFU of virus/cell. EC50 values were 100 to 500 nM for the pre-2004 viruses and in the 5 to 50 nM range for the post-2004 viruses, indicating an increased sensitivity of the more recent viruses to oseltamivir. The good correlation between the oseltamivir IC50 and EC50 was in agreement with a previously published study (34). Notably, however, for the A/Vietnam/1203/04 and A/Hong Kong/156/97 viruses, Yen et al. reported an absence of correlation between the oseltamivir EC50 and the dose of oseltamivir required for an antiviral effect in a mouse model (34). These observations underscore the need for a better understanding of the host and viral factors that affect the in vivo efficacy of oseltamivir treatment, such as the host response, the viral replicative efficiency and tissue tropism, and the hemagglutinin (HA)-NA balance. The inhibition of NA activity can be compensated for by mutations in the HA protein which reduce the HA binding efficiency and restore a functional balance between the receptor-binding and receptor-destroying activities of the surface glycoproteins (28). Most of the key residues within the receptor binding site are conserved in the H5 sequences of the viruses included in our study (data not shown). Significant substitutions were S227N (H3 numbering) in the A/Hong Kong/213/03 isolate, which has been shown to increase its affinity towards {alpha}2,6 sialic acid analogs and decrease its affinity towards {alpha}2,3 sialic acid analogs (5), and the R216E S221P double substitution in the A/Hong Kong/156/97 isolate, which has been shown to decrease binding to branched fucosylated glycans (24). The presence of a Thr at position 160 (H3 numbering) in the 2004-2005 viruses, in place of an Ala in earlier viruses, results in glycosylation of residue N154 and is predicted to reduce the HA affinity for sialosides (32). The fact that these differences in the HA did not compensate for the increased sensitivity to OC of the neuraminidases of the 2004-2005 viruses in our in vitro MDCK-based virus inhibition assay is not necessarily predictive of the effects of the HA-NA balance on viral replication in vivo. However, the sensitivity of the targeted neuraminidase to the drug remains a key parameter of antiviral treatment definition (see Appendix). Our observations call for further investigation of the clinical relevance of a 10-fold increase in sensitivity of the NA to oseltamivir. Indeed, the clinical effectiveness of antiviral treatment could become a major public health concern in case of a pandemic, when optimal utilization of limited stocks of antivirals will be required.

It is already described that upon treatment, oseltamivir-resistant influenza viruses with a >300-fold decrease in sensitivity to the drug can be selected (12, 13). Importantly, our data point to the fact that natural variation in the absence of any drug selective pressure may result in less pronounced but significant variations in the sensitivity of influenza viruses to anti-NA drugs. Whereas a 10-fold increase in sensitivity, as shown here, would not have a negative impact on treatment efficacy, a 10-fold decrease in sensitivity could compromise the treatment efficacy at the commonly used dosage and could favor the emergence of fully resistant viruses. Therefore, in addition to monitoring of the emergence of drug-resistant viruses in patients under treatment, systematic evaluation of the impact of natural genetic variation on the sensitivity to anti-NA drugs should be performed, especially for rapidly evolving, potentially pandemic influenza viruses.


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APPENDIX
 
The reduction of neuraminidase activity in the presence of a competitive drug is predicted by the following equation:

Formula
where vi is the rate of substrate hydrolysis in the presence of inhibitor, v0 is the rate of substrate hydrolysis in the absence of inhibitor, [S] is the substrate concentration, Km is the Michaelis-Menten constant for the hydrolysis of sialic acid by influenza neuraminidase, [I] is the inhibitor concentration, and Ki is the dissociation constant for the enzyme-inhibitor complex. Thus, to achieve a given level of inhibition of a neuraminidase with a fivefold lower Ki, a fivefold lower concentration of inhibitor is required.


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ADDENDUM IN PROOF
 
Shortly after acceptance of this article, the three-dimensional structure of the neuraminidase of an avian H5N1 influenza virus was published (R. J. Russell, L. F. Haire, D. J. Stevens, P. J. Collins, Y. P. Lin, G. M. Blackburn, A. J. Hay, S. J. Gamblin, and J. J. Skehel, Nature 443:45-49, 2006). The tyrosine at position 252 was shown to be involved in a network of hydrogen bonds and to contribute to resistance to oseltamivir of N1 neuraminidases with the His274Tyr substitution. These data are in agreement with our hypothesis that the presence of a histidine at position 252 in some recent H5N1 isolates could be responsible for the increased affinity of their NA for oseltamivir carboxylate.


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ACKNOWLEDGMENTS
 
We are very grateful to Alan Hay, Wilina Lim, and Masato Tashiro for providing influenza A (H5N1) virus isolates and to Mai Le and Jean-Marc Reynes for providing clinical specimens. We thank Monica Marasescu, Saliha Azebi, Frédérique Cuvelier, Patricia Jeannin, and Vanessa Liot for technical assistance. The expert advice of Sophie Guillot for phylogenetic analysis is gratefully acknowledged. We also thank Paul Welti and Jeanne Chiaravalli for helpful suggestions.

This work was supported in part by the viRgil (European Vigilance Network for the Management of Antiviral Drug Resistance [LSHM-CT-2004-503359]) program. M. A. Rameix-Welti was supported by a fellowship from the Comité des Maladies Infectieuses et Tropicales (Institut Pasteur).


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FOOTNOTES
 
* Corresponding author. Mailing address: Unité de Génétique Moléculaire des Virus Respiratoires, URA CNRS 1966, Institut Pasteur, 25-28 rue du Docteur Roux, 75015 Paris, France. Phone: 33-1-45 68 88 11. Fax: 33-1-40 61 32 41. E-mail: nnaffakh{at}pasteur.fr. Back

{triangledown} Published ahead of print on 28 August 2006. Back


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Antimicrobial Agents and Chemotherapy, November 2006, p. 3809-3815, Vol. 50, No. 11
0066-4804/06/$08.00+0     doi:10.1128/AAC.00645-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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