NISN was established to monitor antiviral susceptibility and the potential public health impact of clinical use of the NAIs (34
). To establish a baseline level of susceptibility of circulating wild-type strains and the incidence of primary occurrence of resistance during the 3 years before introduction of the drugs, 1,054 viruses representing the 1996 to 1999 influenza seasons were studied for susceptibility to zanamivir and oseltamivir; no extreme outliers were identified (20
). Furthermore, none of the sequenced mild-outlier viruses was found to contain NA substitutions known to confer resistance (20
). The present study used similar methods applied to viruses collected during the first 3-year period of increasing use of the drugs in certain parts of the world following licensure. A significant decrease in susceptibility (>10-fold) to the NAIs, particularly oseltamivir, was detected in eight viruses during screening by chemiluminescence assay of 2,287 unselected clinical isolates, none of which were obtained from individuals known to have had treatment with NAIs. These isolates included those collected from Japan and the United States, the two countries with greatest per capita NAI use during these years. Such observations are consistent with findings that selection of oseltamivir- or zanamivir-resistant mutants is generally slow in the laboratory and that they are often less infectious and transmissible in experimental animal models (2
On sequencing, the NA of only one virus, A/Mississippi/3/2001 (H1N1), had an H274Y mutation previously associated with resistance after oseltamivir treatment (13
). All other viruses had changes not previously seen in isolates from clinical studies or had no sequence changes from the consensus sequence of circulating wild-type strains. Both B/Perth/211/2001 and A/Hokkaido/15/2002 had mixed populations of resistant and sensitive viruses, thus confirming the role of their mutations in resistance. The B/Perth/211/2001 virus had a D198E (D197 B numbering) NA mutation. A mutation at this residue, the D198N mutation, has also been seen in an influenza B virus isolated from an immunocompromised patient treated with oseltamivir (6
). Structurally, residue 198 is adjacent to R152 in the enzyme active site. An R152K mutation is known to confer resistance to both zanamivir and oseltamivir in influenza B NAs (6
). Hence, it is plausible that the D198E mutation may disrupt the interaction of R152 in the active site, leading to altered binding of the inhibitors.
Although the Y155H mutation confers resistance in the A/Hokkaido/15/2002 virus and Y155 is conserved in all human N1 viruses, H155 is also found in some swine and avian N1 viruses and in some earlier N2 viruses. In fact, the NA of reassortant A/NWS/Tokyo/67 (H1N2), on which the design of zanamivir was based, has H155 yet is clearly sensitive to the inhibitors (27
). Interestingly, the NAs of the highly pathogenic avian influenza viruses circulating in Asia also have H155 and yet are also sensitive to the inhibitors (18
). Such a finding reinforces the conclusion from earlier work that resistance motifs are likely to vary between different influenza NA subtypes and even within subtypes.
Three viruses with decreased susceptibility to oseltamivir had mutations in the NA in the highly conserved sequence ILRTQES (residues 222 to 228): B/New York/39/2001 had an I222T mutation, A/Hamamatsu/92/2002 had an I222V mutation, and A/Belgium/969/2002 had a Q226H mutation. A role for these mutations in altered binding of the NAIs is supported by the findings of others where viruses with I222T and I222V NA mutations were generated in N1 and N2 viruses, respectively, after passage in oseltamivir in vitro (Gilead Sciences, data on file). These led to two- and fourfold decreases in sensitivity, respectively. I222 is located in the substrate binding pocket, near the N
-acetyl and glycerol side chains of the ligand. It is also near R224, which is important in the formation of a salt link to E276, which creates the hydrophobic pocket necessary to accommodate the side chain of oseltamivir (26
). While Q226 is below the active site, it is also located under R224. Therefore, both I222 and Q226 may play an important structural role, being needed for the proper orientation of R224 for oseltamivir binding. Hence, mutations at these sites could impact oseltamivir sensitivity.
The A/New York/24/2001 NA differed by two amino acids from NAs of other viruses currently circulating, G248R and I266V. Other viruses in the database have either one of these variations but are sensitive to the inhibitors. Molecular modeling shows that these amino acids are more than 20 Å away from each other. Hence, it is unclear how these variations could have a synergistic effect. However, the role of at least the G248R mutation in altered susceptibility cannot be discounted, since it is adjacent to H274 and an H274Y mutation confers oseltamivir-specific resistance.
Overall, the present results also indicate that resistance may involve amino acid substitutions not previously observed in clinical trials. However, further detailed characterization of plaque progeny of the other extreme-outlier viruses is needed to confirm the role of potential mutations in altered drug sensitivity, and this emphasizes the current level of uncertainty in linking phenotypic data with genotypic information when screening unselected isolates for altered susceptibility to NAIs.
Evidence suggests that the viruses with decreased NAI susceptibility in vitro detected in the current study came from untreated individuals; several were from parts of the world with little use of NAIs. Therefore, a low level of naturally occurring resistant variants may be present. Transmission from an NAI-treated patient remains a possibility especially for the isolate with the known oseltamivir-resistant H274Y mutation. However, recent studies of ferrets indicate that the most commonly observed mutant viruses from clinical trials are less infectious and also may transmit less readily than their wild-type parents (10
). This lack of fitness in some but not all of the known resistant viruses is in sharp contrast to the transmissible nature of viruses resistant to the M2 inhibitors (9
Evaluation of resistance has been an integral part of the prelicensure and other studies of the NAIs. Resistance was not encountered in clinical trials of zanamivir, and the only mutant resistant to that drug was identified in a virus recovered from a highly immunosuppressed child (8
). Variants resistant to oseltamivir have only rarely (<0.5%) been detected in placebo-controlled treatment studies of immunocompetent adults but have been detected in published studies of treatment of children up to a frequency of 4% (28
). This probably reflects the higher level of replication and more prolonged shedding of virus in children experiencing a primary or second infection. Recently, a higher frequency (18%) of detection of oseltamivir-resistant viruses from young children has been reported, when highly sensitive detection techniques were employed (16
). In contrast, similar techniques have identified amantadine-resistant variants in 80% of treated children (23
In much of the world, the NAIs, particularly zanamivir, were little used during this survey. They were introduced into Japan, the country with the current greatest per capita use, only in 2000 to 2001. Questions about the extent of selective pressure on virus evolution towards altered drug susceptibility can be answered only by continued monitoring of viruses obtained mainly from surveillance in parts of the world where usage of the drugs has increased versus parts of the world where it has not. A clear answer has become even more critical with the discovery that the recent type A (H5N1) avian viruses, a potential source of the next pandemic strain, are susceptible only to the NAIs; preliminary data indicating the detection of reduced oseltamivir susceptibility in an H5N1 isolate due to the recognized H274Y substitution recovered from treated human cases of H5N1 emphasize the importance of this work (4
). Future NISN work will concentrate on surveillance for drug resistance in clinical isolates of influenza obtained in areas of the world with high drug usage and attempt to better understand the relationship between phenotype and genotype with respect to altered susceptibility to NAIs.