Download Characterization of 2 Influenza A(H3N2) Clinical Isolates with

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Characterization of 2 Influenza A(H3N2) Clinical Isolates with Reduced
Susceptibility to Neuraminidase Inhibitors Due to Mutations in the
Hemagglutinin Gene
Yacine Abed,1 Anne-Marie Bourgault,2
Robert J. Fenton,3 Peter J. Morley,3 David Gower,3
Ian J. Owens,3 Margaret Tisdale,3 and Guy Boivin1
1
Research Center in Infectious Diseases of the CHUQ-CHUL
and Laval University, Quebec City, and 2CHUM-St-Luc, Montreal,
Quebec, Canada; 3GlaxoSmithKline, Medicines Research Centre,
Stevenage, United Kingdom
Previous studies have shown that amino acid changes in the hemagglutinin (HA) gene of
influenza viruses may result in decreased susceptibility to neuraminidase inhibitors (NAIs) in
vitro. However, the emergence and characteristics of such HA variants in the clinical setting
remain poorly studied. Herein, we report 2 influenza A(H3N2) isolates, from untreated patients,
harboring an Arg229rIle substitution in the HA1 gene. The Ile229 variants were as sensitive as
the Arg229 viruses to zanamivir and oseltamivir in neuroaminidase inhibition assays but were
significantly less susceptible (by 60–140-fold) in cell-based assays. Although the Ile229 variants
adsorbed less efficiently to Madin-Darby canine kidney (MDCK) cells in kinetic binding assays,
they remained very sensitive to zanamivir in ferrets. Our study shows the importance of the
HA1 229 residue in virus binding to MDCK cells and confirms the unreliability of cell-based
assays in predicting the in vivo susceptibility of HA variants to NAIs.
Influenza virus infections remain a major health problem
worldwide. In addition to immunization programs that use inactivated influenza virus strains, the development of effective
antiviral agents has an enormous potential to control annual
influenza epidemics and future pandemics.
Hemagglutinin (HA) and neuraminidase (NA) proteins of
influenza viruses have crucial functions in the viral life cycle.
HA is responsible for the attachment of the virus to the host
cell surface by binding to sialic acid (SA)–containing oligosaccharide receptors and for subsequent virus penetration into the
cytoplasm through the fusion of endosomal and viral membranes [1, 2]. On the other hand, the main function of NA is
to promote virion release by removing SA residues from viral
glycoproteins and infected cells [3]. NA may also facilitate virus
penetration in the mucin layer of the respiratory tract by allowing virus spread. The catalytic site of the NA enzyme has
been shown to be conserved in all influenza A subtypes and
influenza B viruses [4]. Therefore, NA has been considered as
a suitable target for anti-influenza drugs. Two NA inhibitors
(NAIs), zanamivir and oseltamivir, have been developed [5, 6]
and have now been approved in many countries for the treatReceived 30 January 2002; revised 12 June 2002; electronically published
30 September 2002.
Presented in part: 41st Interscience Conference on Antimicrobial Agents
and Chemotherapy, Chicago, December 2001 (abstract H-661).
Financial support: Canadian Institutes for Health Research and
GlaxoSmithKline Canada (collaborative grant DOP-42568 to G.B.).
Reprints or correspondence: Dr. Guy Boivin, CHUQ-CHUL, Rm. RC709, 2705 Blvd. Laurier, Sainte-Foy, Quebec, Canada G1V 4G2 (Guy
[email protected]).
The Journal of Infectious Diseases 2002; 186:1074–80
䉷 2002 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2002/18608-0003$15.00
ment of influenza virus infections. However, in light of the
experience with other anti-influenza compounds, such as the
adamantanes rimantadine and amantadine, some concerns
have been raised about the potential for the development of
resistance to NAIs [7].
Several in vitro studies have shown that resistance to NAIs
may result from amino acid substitutions in NA, HA, or both
[8–13]. NA changes have been predominantly ascribed to aa
119 and 292, which are part of the framework and catalytic
residues, respectively [4, 9, 11–13]. In addition to the NA mutations, most resistant influenza viruses generated in vitro also
contained mutations in or near the HA receptor–binding site
[10, 12, 13]. In one of these studies, it was demonstrated that
substitution of the HA1 Arg229 residue by a Ser or an Ile
resulted in a significant reduction of virus susceptibility to NAIs
in cell-based assays [10]. The Arg229, which is part of the left
edge of the HA receptor–binding site, is conserved in 12
(H1–H12) influenza A subtypes [14] and seems to be important
for HA stability [10]. We recently isolated 2 influenza A(H3N2)
viruses with an Ile at position 229 of the HA1 subunit from
untreated patients. The aim of the present study was to analyze
the HA properties and the drug phenotype of these HA1 229Ile
variants both in vitro and in ferrets.
Patients, Materials, and Methods
Patients and viruses. Throat and nasal swabs were obtained
from subjects consulting for a flulike illness at a tertiary care center
in Montreal, during the 1999–2000 influenza season. Refrigerated
samples were inoculated within 48 h into roller tubes that contained
primary rhesus monkey kidney cells. Positive cytopathic effects
were confirmed by monoclonal antibody typing and multiplex re-
JID 2002;186 (15 October)
Influenza Virus HA Variants
verse-transcription polymerase chain reaction (RT-PCR) subtyping, as described elsewhere [15], and supernatants from positive
cultures were harvested and stored at ⫺80⬚C for future studies.
NA inhibitors. Zanamivir and oseltamivir carboxylate were
synthesized at the GlaxoSmithKline Medicines Research Centre
(Stevenage, UK).
Sequencing of the HA and NA genes. Viral RNA was isolated
from low-passage culture supernatants using the QIAamp Viral
RNA kit (Qiagen). Complementary DNA was prepared with specific HA or NA 3 primers [16, 17] using the SuperScript II RT
(Gibco BRL), according to the manufacturer’s instructions. PCR
was performed using the Pfu turbo polymerase (Stratagene) in
standard conditions with HA- or NA-specific primers [16, 17]. Cycling conditions were as follows: an initial denaturation step at
95⬚C for 3 min, followed by 35 cycles of 94⬚C for 1 min, 50⬚C for
1 min, and 72⬚C for 3.5 min. The reaction ended with a final
elongation step of 7 min at 72⬚C. PCR products were run on a
1.2% agarose gel and purified using a QIAquick gel extraction kit
(Qiagen). Purified fragments were then sequenced in an automated
DNA sequencer (ABI Prism 377 DNA sequencer; Perkin Elmer)
with the same primers used for PCR amplification.
Kinetics of binding in plaque assay. The kinetics of virus binding
in the plaque assay were analyzed as described elsewhere [13]. Confluent monolayers of MDCK cells in 6-well plates were washed,
and ∼100 pfu of virus in 0.6 mL of Eagle’s MEM that contained
2 mg/mL tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)
trypsin were allowed to adsorb at room temperature. The medium
was removed after different adsorption times (15, 30, and 60 min),
then 3 mL of a 0.6% agar overlay in MEM that contained 0.2%
bovine serum albumin, 2 mg mL/TPCK, and 0.001% DEAE dextran
was added. After a 3-day incubation at 37⬚C, the agarose overlay
was discarded and plaques were counted after staining with 0.1%
crystal violet that contained 10% formaldehyde. The number of
plaques generated after 15 and 30 min of adsorption was compared
with that obtained after a standard adsorption time (60 min).
Hemagglutination and hemagglutination-elution assays.
Hemagglutination assays were performed in U-bottom microtiter
plates using 50 mL of a 1% suspension of untreated red blood cells
from humans, chickens, and horses, as well as human red blood
cells treated with the a2,3-sialidase from Salmonella typhimurium
LT2 (New England Biolabs) [18] and 50 mL of serial 2-fold dilutions
of virus in PBS. Plates were incubated for 1 h at 4⬚C. Hemagglutination-elution assays were performed as described elsewhere [10].
In brief, 8 HA units were preincubated for 30 min at room temperature with either no drug or with serial 2-fold concentrations
of zanamivir ranging from 1 to 0.03 mM (final concentration). Human red blood cells were then added, and the virus was allowed
to agglutinate at 4⬚C for 1 h. Plates were then incubated at 37⬚C,
at which temperature the NA is active, and the elution was followed
by the appearance of pelleted erythrocytes. The concentration of
drug at which virus still eluted was recorded.
NA enzyme–inhibition assay. NA activity was evaluated using
a chemiluminescence-based assay that uses a 1,2-dioxetane derivative of SA as the substrate [19]. NA activity of influenza virus
isolates was first titrated by serial 2-fold dilutions of the virus, and
then NA inhibition was evaluated in the presence of serial 3-fold
concentrations of NAIs ranging from 0.025 to 1.5 mM.
MTT assay. Growth inhibition of influenza virus isolates in
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MDCK cells was performed using a colorimetric method based on
the in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by viable cells, as described elsewhere
[20]. The percentage of cell survival was calculated using the formula [(Ai ⫺ A 0i)/(Ac ⫺ A 0i)] ⫻ 100, where Ai is the absorbance at a
certain NAI concentration, A0i is the absorbance with no drug, and
Ac is the absorbance with no virus. The IC50 values were calculated
by plotting cell survival (percentage) versus drug concentrations.
Influenza virus infection in ferrets. Groups of 4 female ferrets
(weighing 900–1500 g) per treatment regimen were infected by intranasal (inl) instillation of 250 mL of a single influenza virus isolate
that contained 103 pfu/mL while under light anesthesia (isoflurane).
Ferrets received 2 prophylactic doses of zanamivir, at 26 and 2 h
prior to infection, and were treated at 5 h after infection and then
twice daily for 5 days. Animals were weighed daily for 9 days, and
inl doses of zanamivir (1, 0.1, or 0.01 mg/kg of body weight, calculated daily according to animal weight) were administered in a
volume of 0.25 mL/kg. Virus-infected control animals were sham
dosed with pyrogen-free MilliQ PBS only.
Temperature profiles of ferrets were recorded every 10 min with
implanted telemetric transmitters (Dataquest; Data Sciences) prior
and ⭓9 days after infection. Body temperature areas under the
curve (AUCs) were calculated for the period of pyrexic response
(0–96 h postinfection); AUCs were computed as the area above
and below the preinfection mean. Pyrexia was defined as the elevation of core body temperature 12 SD above a preinfection mean
temperature for a period of at least 12 h during the postinfection
period.
Nasal washings were taken from ferrets on a daily basis, prior
to dosing with the compound, by the instillation of 5 mL of PBS
into the external nares of fully conscious ferrets. Expelled PBS was
collected in a petri dish, observed, and subjectively scored for turbidity on a scale of 0–4 (0, clear; 4, thick nasal exudate). The virus
titers in nasal-washing samples after challenge were determined by
an ELISA procedure that used an isolate-specific ferret antiserum,
as described elsewhere [21]. Each ferret nasal-wash sample was
assayed in triplicate at 8 dilutions, and the TCID50 values were
calculated using the Reed-Muench method. The AUCs for the
nasal-wash virus titers on days 1–4 were calculated. As a measure
of the activity of zanamivir, the AUC10 was determined, which is
the dose required to reduce the mean log virus titer to 10% of that
of vehicle-treated animals.
Results
Sequences of the NA and HA genes. The nucleotide sequences of the HA1 and NA genes of various clinical influenza
virus isolates were determined for epidemiological purposes
[22]. As shown in table 1, changes were detected in the HA
receptor–binding site (aa 226; H3 numbering) and in the left
edge of this pocket (aa 229; H3 numbering) in isolates HA61
and HA103, compared with the other Sydney/5/97-like viruses
represented by isolate HA49. In Sydney/5/97-like viruses, the
amino acids at positions 226 and 229 are Ile and Arg, respectively, whereas isolates HA61 and HA103 had, respectively, Val
and Ile at these positions. The isolate HA50 is 226V but has
an Arg residue at position 229. The frequency of isolation of
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Abed et al.
Table 1.
Hemagglutination-elution properties and kinetics of adsorption of clinical HA1 influenza virus variants.
hRBCs
thRBCs
eRBCs
cRBCs
Elution
in absence
of drug, h
64
128
64
64
ND
64
16
16
ND
0
0
0
ND
0
0
0
18
18
1
1
a
HA1 change
Isolate
Nucleotide
b
676A–686G
676G–686G
676G–686T
676G–686T
HA49
HA50
HA61
HA103
JID 2002;186 (15 October)
Amino acid
226I–229R
226V–229R
226V–229I
226V–229I
HA titer
Highest
zanamivir
concentration
allowing virus
elution, mM
60 min
30 min
15 min
0.03
0.03
11
11
100
ND
100
ND
75
ND
45
ND
60
ND
20
ND
Plaques in MDCK cells
after adsorption times, %
NOTE. HA, hemagglutinin; ND, not determined.
a
HA titers were determined by standard hemagglutination assays using human red blood cells (hRBCs), a2,3-sialidase–treated human RBCs (thRBCs), equine
RBCs (eRBCs), and chicken RBCs (cRBCs).
b
Wild type compared with A/Sydney/5/97 and A/Wuhan/359/95, at codons 226 and 229.
the R229I mutation was ∼4.4% for H3N2 isolates collected in
the Province of Quebec from 1997–2000, whereas most (97.7%)
of the isolates were 226V. The NA catalytic region, consisting
of 8 framework and 10 functional residues [4], was conserved
in all our isolates.
Kinetics of virus binding. The binding affinity to MDCK
cells was found to be lower for isolate HA61 than for isolate
HA49. As shown in table 1, a reduction in the adsorption time
from 1 h to 15 min more significantly affected the virus yield
(plaque nos.) obtained with isolate HA61 than that obtained
with isolate HA49. The virus yield obtained after 15 min of
adsorption corresponded to 20% of that generated after 60 min
of adsorption for isolate HA61, whereas the percentage was
60% for isolate HA49.
Hemagglutination and hemagglutination-elution properties.
All tested viruses agglutinated untreated and a2,3-sialidase–
treated human red blood cells, whereas none of the latter was
able to agglutinate chicken or equine red blood cells (table 1).
The HA titers of the R229I variants decreased by a factor of
4 when the treated human red blood cells were used compared
with untreated human cells. For hemagglutination-elution analysis, viruses were allowed to agglutinate human red blood cells
at 4⬚C for 1 h, then the plates were incubated at 37⬚C, which
allowed NA activity to elute virus from agglutinated cells. As
shown in table 1, in the absence of zanamivir, complete elution
was seen after 1 h of incubation at 37⬚C for isolates HA61 and
HA103. In contrast, an 18-h period was necessary to elute isolates HA49 and HA50. After prior incubation with zanamivir,
the highest concentration at which isolates HA49 and HA50
eluted was 0.03 mM, whereas isolates HA61 and HA103 still
eluted at the highest concentration used in the assay (1 mM).
Susceptibility to NAIs determined by the NA enzyme-inhibition
assay. The results of the NA inhibition assays for the different
HA variants are shown in table 2. Both zanamivir and oseltamivir inhibited the enzyme activity of the 4 isolates at a similar
level. The IC50 values ranged from 2.5 to 4.25 nM in the case
of zanamivir and from 2.3 to 4.25 nM for oseltamivir. These
IC50 values are within the range of those obtained for other
wild-type H3N2 isolates tested in our laboratory (data not
shown).
Susceptibility to NAIs determined by the MTT assay. As
shown in table 2, significant differences were obtained in viral
growth inhibition using the MTT assay when comparing isolates HA49 and HA50 on one side and isolates HA61 and
HA103 on the other. Using this method, the IC50 values of
isolates HA49 and HA50 for the 2 drugs were !4 nM. By
contrast, the IC50 values for isolates HA61 and HA103 were
60–140-fold higher, ranging from 175 to 180 nM for zanamivir
and from 356 to 429 nM for oseltamivir. These results demonstrate that the change at residue 229 was entirely responsible
for the altered drug phenotype in cell-based assays.
Virulence and zanamivir susceptibility of the Ile229 variant in
the ferret model. Because the altered HA properties of our
variants were ascribed to the change at residue 229, the isolate
HA61 was selected for in vivo experiments. The virulence of
this variant was first evaluated in ferrets by recording the pyrexic response of infected animals. All 4 vehicle-treated control
animals infected with isolate HA61 had a pyrexic response between 2 and 4 days after infection, with a peak at 60 h (figure
1A). In contrast, the pyrexic responses in zanamivir-treated
ferrets were completely suppressed in all animals at the minimal
dose of 0.01 mg/kg (figure 1B).
Analysis of body weight changes revealed that vehicle-treated
control animals had a mean body weight loss of ∼7% at day 9
after infection, reflecting of reduced food and water consumption.
In contrast, the mean body weight of animals treated with zanamivir (0.01–1 mg/kg) remained similar throughout the course
of the study (data not shown). Moreover, at all doses of zanamivir, there was a clear reduction in nasal-wash turbidity scores,
compared with vehicle-treated control animals (data not shown).
The antiviral activity of zanamivir, when given inl, at doses of
1, 0.1, or 0.01 mg/kg to ferrets infected with isolate HA61 is
shown in figure 2. Zanamivir was effective in reducing the nasalwash viral titers in ferrets at doses as low as 0.01 mg/kg. An
AUC10 was calculated as !0.01 mg/kg, which is comparable to
values obtained for other isolates tested in this model [21].
Discussion
Monitoring of influenza virus susceptibility to the commercially available NAIs (zanamivir and oseltamivir) is an impor-
JID 2002;186 (15 October)
Influenza Virus HA Variants
Table 2. Susceptibility of clinical HA1 influenza virus variants to
neuraminidase (NA) inhibitors as assessed by NA inhibition and MTT
assays.
IC50 value
in NA assay, nM
IC50 value
in MTT assay, nM
Isolate
Zanamivir
Oseltamivir
Zanamivir
Oseltamivir
HA49
HA50
HA61
HA103
3.25
2.50
3.85
4.25
3.00
4.25
2.40
2.30
1.24
2.85
175.76
180.79
2.97
3.84
356.22
429.54
NOTE.
Nos. are the mean of 4 experiments. HA, hemagglutinin.
tant clinical task [23, 24]. The study of drug-resistance mechanisms in clinical influenza virus isolates is of particular interest
[25, 26].
In the present study, we performed a comprehensive characterization of 2 influenza A(H3N2) isolates from untreated
patients containing a 229ArgrIle change in the HA1 gene. Although the 229Ile variant was associated with reduced binding
affinity to MDCK cell receptors and decreased susceptibility
to NAIs in cell-based assays, our study shows that this virus
was highly virulent and very susceptible to zanamivir in ferrets.
It appears from in vitro studies that, besides the single NA
and double HA/NA mutants, viruses harboring unique mutations in some regions of the HA gene may have a decreased
binding affinity to cell receptors with a reduced susceptibility
to NAIs [10]. However, the clinical impact of such HA variants
remains to be determined [23]. Moreover, there is no evidence
at present that the R229I mutation reported in our study could
be induced by the use of NAIs in humans, because such a
mutation was not found in our previous study that investigated
the antiviral effects of zanamivir [27].
The left edge of the influenza virus HA receptor-binding pocket
is one of the determinants of receptor specificity [28–31]. A variant with a deletion of 7 amino acids (224–230) in the HA1
subunit exhibited reduced adsorption and only transient hemagglutination activity [32]. aa 226 is of particular importance in
receptor-binding specificity [29, 33]. Residue 229 is one of highly
conserved residues in the left edge of the binding pocket, which
suggests a possible role for this residue in the HA structure and/
or function [14]. Two H1N9 variants with substitutions at position 229 have been generated after in vitro passages in the
presence of the NAIs zanamivir and 4-amino-Neu5Ac2en. One
variant had a Ser, whereas the other had an Ile residue at this
position and contained an additional 223ValrIle mutation [10].
Of interest, these mutants were ∼100–1000-fold less susceptible
to both drugs tested in cell (MDCK)–based assays [10]. This high
level of resistance to NAIs was attributed to substitutions at the
HA1 229 residue, because sequence analysis did not reveal any
other significant changes in the NA gene.
Our two 229Ile variants (isolates HA61 and HA103) exhibited reduced susceptibility (60–140-fold) to NAIs in cell
(MDCK)–based assays. In addition, these variants were shown
to bind weakly to MDCK cells in the kinetic adsorption assay
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as well as to human red blood cells in the hemagglutinationelution assay, confirming the role of the HA1 229 residue in
cell-binding affinity. However, in contrast to the HA mutants
described by McKimm-Breschkin et al. [10], our HA 229Ile
variants did not exhibit a thermolability or a drug-dependent
phenotype (data not shown). Our results demonstrate that the
Val residue at position 226, which has been present in most
H3N2 viruses isolated after the 1997–1998 influenza season [22],
does not appear to contribute to the reduced susceptibility phenotype. Indeed, isolate HA50, which harbored only the 226Val
mutation, was as sensitive as the Sydney-like isolate HA49.
Residue 226 is important for the binding specificity and hostrange restriction of influenza viruses [28–31], but mutations at
this position have not been reported to alter the susceptibility
to NAIs.
The ferret has proved to be a suitable animal model for
studying the pathogenicity of influenza virus strains and their
susceptibility to antiviral agents [34–36]. In the present study,
the virulence and the zanamivir susceptibility of variant 229Ile
were evaluated in ferrets after inl challenge. The variant was
first found to be highly virulent, inducing strong pyrexic responses and nasal discharge. Inl doses of zanamivir as low as
0.01 mg/kg clearly reduced the virus load of the 229Ile isolate
in nasal-wash samples. In addition, this dose reduced nasalwash turbidity, prevented body weight loss, and abolished pyrexic responses, compared with vehicle-treated control animals.
Such results are in agreement with zanamivir-susceptibility results obtained from clinical isolates containing an Arg at position 229 when evaluated in the same animal model [21]. Thus,
our results indicate that the influenza virus variant 229Ile is
virulent and causes disease in the ferret. This variant was highly
susceptible to inhibition by zanamivir compared with both susceptible laboratory strains and clinical isolates of influenza A
and B viruses [21, 23], which is in contrast to in vitro data.
The discrepancy between the phenotypes of the 229Ile variant
as determined in vivo and in the MDCK-based assay could be
attributable to differences in cell receptors expressed in the 2
systems. Human influenza viruses bind preferentially to the
Neu5Ac(a2,6)Gal-terminated receptors and have a reduced affinity for those harboring the Neu5Ac(a2,3)Gal [37, 38]. As in
ciliated cells of the human airways, the ferret airway cells have
receptors that contain the SA a2,6 linkage [35]. In contrast,
MDCK cells express receptors of both SA linkage types, but
the SA a2,3 is predominant [37]. Thus, HA mutations resulting
in decreased binding affinity for one cell receptor type may not
alter the affinity for other receptor types.
The difference between the 2 receptor systems may yield
aberrant results [39, 40]. The substitution of HA residue 198
(198ThrrIle) in an influenza B clinical isolate resulted in a
reduced viral affinity for human cell receptors and a concomitant increase in affinity for MDCK cell receptors [39]. This
increased affinity for MDCK receptors masked the effect of
the NA substitution (152ArgrLys) and led to a zanamivir-
Figure 1.
Core body temperatures in ferrets infected with influenza isolate HA61 and sham-treated with intranasal doses of PBS (A) or treated with 0.01-mg/kg intranasal doses of zanamivir (B)
JID 2002;186 (15 October)
Influenza Virus HA Variants
Figure 2. Virus titers in ferrets (n p 4) infected with influenza virus
isolate HA61 and treated with intranasal doses of zanamivir (ZMV)
or vehicle.
1079
In summary, our R229I variants appear to have reduced
affinity for both cell receptor types (a2,3 and a2,6), as shown
by a lower binding affinity to MDCK cells on one side and,
on the other, rapid elution from human red blood cells together
with lower HA titers when a2,3 sialidase–treated human red
blood cells are used. However, such results do not explain why
viral susceptibility to NAIs was unaffected in ferrets that were
reported to harbor predominantly SA a2,6 Gal receptors. It is
possible that the reduction in viral susceptibility in the ferret
may have been too subtle to detect, although further investigations are needed to confirm this hypothesis.
Altogether, these data confirm the unreliability of the MDCK
cell–culture system for predicting resistance to NAIs where an
HA mutation is present. Thus, in the absence of a cell line
carrying the SA receptors reflective of the human respiratory
tract, the NA enzyme–inhibition assay continues to be the most
reliable system for monitoring susceptibility to NAIs. Additional testing of HA variants arising during therapy with NAIs
is warranted to evaluate the role and importance of NA-independent mechanisms of resistance.
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