JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 3517–3524
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 10
Highly Sensitive and Quantitative Detection of the H274Y Oseltamivir
Resistance Mutation in Seasonal A/H1N1 Influenza Virus?
Darwin J. Operario,1Michael J. Moser,2† and Kirsten St. George1*
Laboratory of Viral Diseases, Wadsworth Center, New York State Department of Health, Albany, New York,1and
EraGen BioSciences, Madison, Wisconsin2
Received 21 May 2010/Returned for modification 6 July 2010/Accepted 21 July 2010
A C-to-T transition mutation in the neuraminidase gene from seasonal A/H1N1 causes a His-to-Tyr mutation
at amino acid position 275 (H274Y, universal N2 numbering), conferring resistance against oseltamivir
(Tamiflu). This mutation was first detected in clinical samples in Europe during the 2007-2008 influenza
season. Viruses with this mutation reached a prevalence of ?11% by the end of the season in North American
isolates tested by the CDC. We developed a highly sensitive and specific quantitative real-time reverse
transcriptase PCR assay to detect the H274Y mutation. This assay utilizes a 5?-methyl-isocytosine (isoC)
residue and fluorescent reporters on genotype-specific primers. During PCR, a quencher coupled to isoguanine
(isoG) is site-specifically incorporated complementary to the isoC/dye, resulting in loss of fluorescence.
Optimization of primers and assay conditions produced a limit of detection of 100 gene copies per reaction for
both wild-type and H274Y genotypes. In samples with mixed populations, it can reliably detect as little as a 1%
wild-type or 0.1% H274Y component. This high sensitivity makes the assay usable on samples with viral loads
too low for dideoxy or pyrosequencing analysis. Additionally, the assay distinguishes seasonal A/H1N1 from
A/H3N2, influenza B, or 2009 pandemic A/H1N1, making it useful for influenza virus subtyping as well as for
drug resistance detection. We probed seasonal A/H1N1 samples from the 2005-2006, 2006-2007, and 2007-2008
influenza seasons. Data from the new assay closely matched available drug resistance genotype data previously
determined by dideoxy sequencing. The H274Y mutation was only found in samples from the 2007-2008 season.
Influenza viruses cause considerable annual worldwide mor-
bidity and mortality. In the United States alone, greater than
200,000 persons are hospitalized each year due to influenza
and approximately 36,000 die from influenza-related disease
(16). Vaccination is considered the first and best defense
against influenza. However, the efficacy of protection con-
ferred by annual vaccination can be limited by the strength of
the antigenic match of vaccine strains to the circulating strains.
In addition, herd (community-level) immunity is limited by less
than 100% vaccine coverage. These circumstances allow influ-
enza to easily spread among susceptible persons and through
populations. Antiviral drug treatment and prophylaxis are ad-
ditional and necessary modes of defense against morbidity,
mortality, and further spread of the virus.
Widespread resistance against the adamantane class of drugs
among A/H3N2 viruses, beginning in the 2003-2004 season,
prompted public health officials in the United States to recom-
mend against the use of these drugs during the 2005-2006
season in favor of neuraminidase inhibitors (NAIs) (3). First
approved for clinical use in the United States in 1999 (17),
NAIs target the viral surface protein neuraminidase and are
effective against both influenza A and B. There are currently
two FDA-approved drugs in this class: oseltamivir (Tamiflu;
Roche) and zanamivir (Relenza; GlaxoSmithKline). When
given within the first 48 h of a patient becoming symptomatic,
NAIs have been shown to reduce the duration and severity of
influenza illness in both adults and children (11, 25).
In January 2008, nine European countries reported seasonal
influenza A/H1N1 isolates showing resistance to oseltamivir
(13, 25). In the following months, additional countries, includ-
ing the United States, reported oseltamivir-resistant influenza
viruses (24). These findings were alarming because drug resis-
tance testing during the previous influenza season (2006-2007)
had revealed no oseltamivir resistance in Europe (13). Fewer
than 1% of North American seasonal A/H1N1 viruses from the
same time period tested by the Centers for Disease Control
and Prevention (CDC) showed resistance (4). In addition, dur-
ing clinical trials with oseltamivir, shedding of drug-resistant
virus was noted at a frequency of only 4% in children and in
?1% in adults (28). Analysis revealed that all resistant viruses
were seasonal A/H1N1, carrying the same C3T transition
mutation in the neuraminidase gene, with a resulting histidine-
to-tyrosine change at amino acid position 275 (“H274Y” in
universal N2 numbering). By the end of the 2007-2008 season,
the CDC reported this mutation in 111 of 1,020 tested seasonal
A/H1N1 isolates; 4 were found in New York State (4). Osel-
tamivir-resistant seasonal A/H1N1 spread extensively, becom-
ing the dominant variant in Oceania and Southeast Asia in
May 2008 (10) and with virtually all seasonal A/H1N1 strains
possessing the H274Y mutation during the 2008-2009 influ-
enza season in the United States (5).
The need for continual monitoring for antiviral drug resis-
tance among influenza viruses is highlighted by several factors.
Use of antiviral medications as a treatment and prophylactic is
an integral component of infection control during influenza
outbreaks. With the advent of adamantane resistance, use of
* Corresponding author. Mailing address: David Axelrod Institute,
Wadsworth Center, New York State Department of Health, P.O. Box
22002, Albany, NY 12201-2002. Phone: (518) 402-2709. Fax: (518)
473-4336. E-mail: firstname.lastname@example.org.
† Present address: Lucigen Corporation, 2120 W. Greenview Dr.,
Suite 9, Middleton, WI 53562.
?Published ahead of print on 28 July 2010.
the neuraminidase inhibitors, most commonly oseltamivir,
has risen dramatically. In addition, oseltamivir is a major
component of influenza pandemic preparedness stockpiles
in the United States. Continual improvement of methods for
the survey and detection of oseltamivir resistance in all influ-
enza isolates is imperative to ensure the lasting viability of this
drug as an antiviral treatment.
Laboratory testing for NAI resistance can be performed via
a phenotypic neuraminidase inhibition assay, giving a 50%
inhibitory concentration (IC50) for a drug of interest. Labora-
tories can also use nucleic acid testing such as real-time quan-
titative reverse transcriptase (RT)-PCR as well as dideoxy se-
quencing and pyrosequencing methods. Sequencing methods
provide detailed information about gene regions of interest
and enable the identification of new mutations that may have
an effect on drug resistance. Pyrosequencing, in particular, has
been reported to be capable of detecting minor populations at
concentrations as low as 10% within mixtures of neuramini-
dase gene targets (7, 8). However, both dideoxy sequencing
and pyrosequencing require additional processing steps after
an initial PCR, as well as specialized sequencing instruments,
software, and training. Real-time quantitative RT-PCR pro-
vides a rapid, highly sensitive, and specific alternative to se-
quencing. This method is also capable of detecting targets in
samples with viral loads too low for detection in sequencing
assays. Furthermore, real-time platforms are available from
multiple manufacturers and are more commonplace in clinical
laboratories than sequencing equipment.
Here, we describe the development of a novel, highly
sensitive real-time quantitative RT-PCR assay for the de-
tection of H274Y in seasonal A/H1N1 influenza. The assay
utilizes MultiCode technology, a system that employs an addi-
tional base pair set of 5?-methyl-isocytosine (isoC) and isogua-
nine (isoG) (12, 22). An isoC and isoG can only base pair to
one another, and neither an isoC-G nor a C-isoG bond can
form. In real-time RT-PCR applications, an isoC base is in-
cluded at the 5? end of a primer, directly linked to a fluoro-
phore, such as 6-carboxyfluorescein (FAM) or hexachlorofluo-
rescein (HEX) (Fig. 1). During PCR, a quencher (Dabcyl)
directly linked to an isoG nucleotide is site specifically incor-
porated into the PCR product complementary to the isoC-
fluorophore, resulting in a detectable decrease in fluorescence
(23). Because multiple fluorophores can be used in the same
reaction, multiplexed assays can be designed. In addition, be-
cause the fluorophores are not cleaved from the oligonucleo-
tides, postamplification melt curve analysis is possible, facili-
tating analyses of assay specificity. This PCR technology has
been successfully employed as part of a PCR panel targeting
several respiratory viruses (15), in assays for other viruses
including hepatitis C (20) and HIV (19, 26, 27), and in the
detection of anthrax-related toxin genes (18).
This new assay was developed with analyses for both muta-
tion detection and facilitation of resistance evolution in mind.
To that end, we developed a novel multiplexed assay with high
sensitivity and specificity and with a higher sensitivity for de-
tecting a minor component mutant population compared to
that detectable by pyrosequencing. The assay was applied to a
collection of A/H1N1 influenza-positive samples collected be-
tween 2005 to 2008, in order to determine whether the H274Y
mutation was present in clinical samples prior to its apparent
emergence during the 2007-2008 season.
MATERIALS AND METHODS
Specimens and standard curve construction. A total of 85 primary specimens
and isolates used in this study were collected in New York State during the
2005-2006, 2006-2007, or 2007-2008 influenza season as part of normal surveil-
lance. Only samples that were previously characterized as seasonal A/H1N1
influenza virus by the Wadsworth Center Virus Reference and Surveillance
Laboratory using real-time reverse transcriptase PCR were included in the
present study. For a subset of these samples, the neuraminidase gene had already
been characterized by dideoxy sequencing (14).
Cultured influenza isolates, used in assay development and validation (see
below), were maintained on rhesus monkey kidney cells (ViroMed Laboratories,
Minnetonka, MN, or Diagnostic Hybrids, Athens, OH). Neuraminidase inhibi-
tor-resistant influenza was maintained with oseltamivir carboxylate at a final
concentration of 10 ?g/ml (25.9 ?M; kindly provided by Roche). Virus cultures
were maintained until cytopathic effect was evident throughout the culture and
Nucleic acids were extracted using the QIAcube (Qiagen USA, Valencia, CA)
automated extractor with the Qiagen QIAamp viral RNA extraction kit accord-
ing to manufacturer’s instructions. Influenza A/H1N1 isolates to be used for
controls and standard curve construction were first quantified in a separate
quantitative real-time RT-PCR assay targeting the matrix gene segment. Stan-
FIG. 1. Primer alignment to target sequence. Primers were designed against a consensus sequence of N1 sequences from seasonal A/H1N1-
positive clinical samples submitted for testing to the Wadsworth Center between 2004 and 2008. The genotype-specific primers are designed in two
sections: one section anneals to the coding section of the N1 gene, and there is a noncoding tail which includes an isoC residue and a fluorophore.
The WT-targeted primer is labeled in HEX, while the mutant-targeted primer is labeled in FAM. The nucleotide at the 3? end of each of the
genotype-specific primers targets the SNP responsible for coding for His or Tyr. A specific primer is used for reverse transcription. The same primer
is paired against the genotype-specific primers during PCR. This primer system creates an amplicon of 66 bp.
3518 OPERARIO ET AL.J. CLIN. MICROBIOL.
dard curves were constructed by testing 10-fold serial dilutions of target, con-
taining between 108and 1 gene copy per reaction. Standard curve reactions were
applied to the multiplexed assay in duplicate, and each test was performed at
least three times.
Primer design. Primers were designed against a database of compiled seasonal
N1 gene sequences derived from New York State samples spanning from 2004 to
2008 and with the HYTHER online nucleic acid hybridization program, acces-
sible at http://ozone3.chem.wayne.edu. The resulting primer designs used in the
final assay are shown in Fig. 1. A specific primer was used for reverse transcrip-
tion. The same primer was also used during PCR and was paired with genotype-
specific primers. The primer targeted against wild-type (WT) N1 was labeled
with HEX, while the H274Y-targeted primer was labeled with FAM. PCRs with
these primers give a 66-bp amplicon.
Real-time reverse transcriptase PCR. The real-time quantitative reverse trans-
criptase PCR was performed with the Plexor one-step RT-PCR system from
Promega (Promega, Madison, WI). The reverse transcription step was per-
formed in a 20-?l volume including Plexor system components, 5 ?l target, and
primer (Fig. 1) to a final concentration of 0.4 ?M, at 45°C for 15 min, followed
by 95°C for 2.5 min. After reverse transcription, FAM (0.4 ?M final concentra-
tion)- and HEX (0.15 ?M final concentration)-labeled primers were added,
giving a final PCR volume of 25 ?l. All PCRs were performed on the Bio-Rad
iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA). The cycling con-
ditions are as follows: 2.5 min at 95°C, 2 cycles of 5 s at 95°C and 35 s at 53°C,
and 45 cycles of 5 s at 95°C and 35 s at 63°C. PCR was followed by melt curve
analysis ranging from 55°C to 95°C, with 30 s per step with a 1°C increase per
step. The iQ5 real-time PCR software is designed to interpret and display data
to the user from reactions that show increases in fluorescence over time. Because
the PCR system used in this study is based upon decreases in fluorescence over
time, data from real-time PCR runs were imported into Promega Plexor soft-
ware, version 1.1.4, for data analysis. When using MultiCode PCR chemistry,
fluorescence is reduced with target amplification and is reported in relative
fluorescence units (RFU). In the MultiCode PCR developed in this study for
resistance mutation analysis, to define a sample as positive for the target, two
criteria must be met: (i) the fluorescence trace on the amplification plot must
cross the (user-defined) threshold line (giving the sample a defined threshold
cycle [CT] value), and (ii) the corresponding trace on the melt curve graph must
show a peak at 73 ? 1°C. As with all real-time PCR assays, CTvalues are
inversely proportional to the target concentration.
Specificity. Specificity was tested by application of the assay to a panel of
nonseasonal A/H1N1 viruses, including the following: influenza B; influenza
A/H3N2; 2009 pandemic A/H1N1; adenoviruses 3, 4, 5, and 8; coxsackieviruses
A9, B3, B4, and B5; human metapneumovirus 1B and 2B; rhinovirus 1A; measles
virus; mumps virus; severe acute respiratory syndrome (SARS) coronavirus;
coronavirus 229E; lymphocytic choriomeningitis virus; enteroviruses 68 and 71;
and echoviruses 6, 9, 11, and 30. Each virus in the panel was tested at a
concentration of at least 106genome copies per reaction, in duplicate. Specificity
experiments were performed at least three times, except for coxsackieviruses A9
and B5, coronavirus 229E, human metapneumovirus 2B, and lymphocytic cho-
riomeningitis virus, which were tested only twice. Seasonal A/H1N1 virus con-
taining WT or H274Y neuraminidase 1 genes was used as the positive control.
We utilized MultiCode technology to detect and quantify
seasonal A/H1N1 clinical samples containing populations of
WT or H274Y mutant neuraminidase genes. Primers were
designed against both the WT and mutant genotypes, such that
the nucleotide at the 3? end of each primer targets the single
nucleotide polymorphism (SNP) coding for either His or Tyr.
The primers are designed in two sections: an annealing seg-
ment that binds the coding region of the gene and a noncoding
tail that includes the isoC nucleotide and fluorophore. As
shown in Fig. 1, the neuraminidase-specific annealing region of
each primer has an annealing temperature of approximately
55°C. The 6-base noncoding tails increase the overall annealing
temperature by approximately 10°C, giving the primers a final
melting temperature (Tm) of approximately 65°C. The WT-
and H274Y-specific primers possess different tail sequences
and fluorophores. This overall primer design allows a two-
stage PCR cycling scheme (see Materials and Methods), which
begins with a lower-specificity cycling Tmof 55°C, followed by
additional cycles with higher specificity annealing/extension at
The assay employs a specific reverse transcription primer,
targeted against the negative-stranded viral RNA. The same
primer is also used during PCR, paired against both the WT-
and H274Y-specific primers. These primers yield a 66-bp am-
plicon on a seasonal N1 target. Melt curve analysis of ampli-
cons generated with either the WT- or H274Y-targeted primer
gives a product with a Tmof 73°C, as shown in Fig. 2.
Limit of detection. Optimization of primer concentrations
and cycling conditions under multiplexed conditions resulted
in a lower limit of detection of 100 WT or H274Y gene copies
per reaction. The limit of detection was determined using
standard curves constructed from quantified influenza virus
extracts from cultured isolates containing pure populations of
either WT neuraminidase or neuraminidase with the H274Y
mutation, as shown in Fig. 2. Some cross-reactivity of the
H274Y primer with WT template was observed at concentra-
tions of ?105WT gene copies. However, such cross-reactivity
is distinguishable due to the presence of a secondary melt peak
at 67°C, as shown in Fig. 2. When such cross-reactivity occurs
on samples with a pure WT neuraminidase genotype, the melt
peak at 67°C is stronger than the peak at 73°C.
Detection of mixed populations. Due to multiplexing, the as-
say is capable of detecting mixed populations within the same
reaction. To test assay sensitivity for mixed populations, quan-
tified WT and H274Y viral nucleic acid extracts were combined
at 1%, 5%, 50%, 95%, and 99% WT mixtures, with a total 106
gene copies per reaction. Additionally, 0.1% and 99.9% WT
mixtures were created, with 105, 106, and 107total gene copies.
The duplexed assay is capable of detecting a WT component as
low as 1% (104gene copies) in an otherwise mutant back-
ground (9.9 ? 105gene copies), as shown in Fig. 3A. As
demonstrated in Fig. 3C, the assay is also capable of detecting
a mutant population as low as 0.1% in an otherwise WT back-
ground containing 107, 106, or 105total gene copies.
Assay specificity. To test assay specificity for seasonal A/H1N1,
the assay was applied to a panel of nucleic acid extracts from
influenza and noninfluenza viruses. Figure 4 shows negative
signal results from a representative experiment. As can be seen
in this figure, there is a limited amount of nonspecific ampli-
fication for nonseasonal A/H1N1 samples, including those with
adenoviruses 4 and 8, coxsackievirus B3, enterovirus 68, and
A/H3N2 influenza virus. However, these nonspecific amplifi-
cations are easily distinguished from specific PCR products
generated on an N1 template, by melt curve analysis. The
primers used in this assay do not strongly cross-react with the
2009 pandemic A/H1N1 target, which can be attributed to
mismatches between the primers and the 2009 pandemic virus
N1 target. Analysis of the primer sequences reveals 11 mis-
matches between the reverse transcription primer and the 2009
pandemic virus N1 sequence and 2 mismatches for each of the
Analysis of archived influenza samples. The H274Y muta-
tion was first detected during the 2007-2008 influenza season,
but we were interested in investigating whether the mutation
had gone undetected in seasonal A/H1N1 samples collected
prior to the 2007-2008 season. A total of 85 archived respira-
VOL. 48, 2010H274Y MUTATION DETECTION USING isoC/isoG TECHNOLOGY3519
FIG. 2. Limit of detection. The lower limit of detection was determined by application of the assay to standard curves from quantified nucleic
acid extracts to give between 108and 1 gene copy per reaction. Standard curve samples were derived from cultures containing seasonal A/H1N1
cultures with either pure WT or pure H274Y neuraminidase genes. The limit of detection for both the WT- and H274Y-targeted primers is 100
gene copies (gc), using the reaction conditions stated in Materials and Methods. In the amplification plots, data from a sample positive for the N1
target is shown by a loss of fluorescence (i.e., decreasing relative fluorescence units [RFU]) over increasing PCR cycles. In melt curve analysis, a
sample positive for an N1 target gives a melt peak at 73°C. Cross-reactivity of H274Y primer against a WT target gives a secondary melt peak at
67°C. (A) WT primer; (B) H274Y primer. Blue, amplification on a WT target; green, amplification on a mutant target; red, no-template control.
3520 OPERARIO ET AL.J. CLIN. MICROBIOL.
tory samples positive for seasonal A/H1N1 were identified for
testing with the new assay. These samples had been submitted
to the Wadsworth Center for testing during the 2005-2006,
2006-2007, and 2007-2008 influenza seasons. Available dideoxy
sequencing data on the N1 gene sequences from 2006-2007 (17
specimens) and 2007-2008 (44 specimens) seasonal A/H1N1-
positive samples (14) were used as a basis for comparison to
the data generated from the MultiCode-based assay.
Results from the new assay indicated that the 44 specimens
from the 2007-2008 season contained either a 100% WT or
100% H274Y neuraminidase genotype. As shown in Table 1,
the data generated by the new assay on samples from the
2007-2008 season closely matched available sequencing data.
Importantly, the four samples that had previously tested pos-
itive for the H274Y oseltamivir resistance mutation by dideoxy
sequencing also tested positive for the mutation by the new
assay. The new assay was additionally able to identify as WT
one sample that dideoxy sequencing had failed to characterize
at all. A total of 41 additional seasonal A/H1N1-positive re-
spiratory specimens from the 2005-2006 (4 specimens) and
2006-2007 (37 specimens) seasons were retrieved from sample
archives at the Wadsworth Center. The new assay detected
only WT neuraminidase in all samples from these seasons. No
samples of mixed genotype were found in samples from any of
the three seasons.
The adamantanes have been approved as antiinfluenza drugs
since 1966 (1) and were commonly prescribed prior to recom-
mendations against their use during the 2005-2006 season.
Amino acid changes in the M2 protein provide resistance to
these drugs and have been reported to emerge within as little
as 6 days after the initiation of rimantadine prophylaxis (2, 9,
21). Resistance to the NAI oseltamivir was first detected in the
2007-2008 influenza season among seasonal A/H1N1 viruses,
all with the H274Y neuraminidase mutation. Viruses possess-
ing the H274Y mutation spread rapidly, even in the absence of
drug pressure, and oseltamivir-resistant seasonal A/H1N1 be-
came the dominant virus of this subtype during the 2008-2009
season. In addition, the H274Y mutation in seasonal A/H1N1
is the equivalent of the H275Y mutation detected in some 2009
pandemic A/H1N1 viruses.
Common molecular methodologies for identifying influenza
virus and detecting drug-resistant strains include dideoxy se-
quencing, pyrosequencing, and quantitative real-time PCR.
Used as part of quantitative reverse transcriptase PCR, the
FIG. 3. Mixed-sample analysis. For panels A and B, nucleic acid
extracts from pure populations of WT and H274Y viruses were mixed
in ratios of 1%, 5%, 50%, 95%, and 100% WT with 106gene copies in
total and applied to the multiplexed assay. Black, 1% WT; orange, 5%
WT; brown, 50% WT; green, 95% WT; blue, 99% WT; red, no-
template control. In panel C, nucleic acid extracts from pure popula-
tions of WT and H274Y viruses were mixed in a ratio of 99.9% major
component to 0.1% minor component genotype with 107, 106, or 105
total gene copies (gc) applied to the multiplexed assay. Purple, 107
gc total; pink, 106gc total; orange, 105gc total; red, no-template
control. Data from these plots are interpreted as described in the
legend to Fig. 2.
VOL. 48, 2010 H274Y MUTATION DETECTION USING isoC/isoG TECHNOLOGY3521
FIG. 4. Specificity analysis. Nucleic acid extracts from a panel of viruses were tested against the assay. A representative experiment is pictured
above. The following viruses are represented in these plots: WT and H274Y seasonal A/H1N1; seasonal A/H3N2; influenza B; 2009 pandemic
A/H1N1; adenoviruses 3, 4, 5, and 8; coronavirus 229E; SARS coronavirus; coxsackieviruses A9, B3, B4, and B5; echoviruses 6, 9, 11, and 30;
enteroviruses 68 and 71; human metapneumoviruses 1B and 2B; lymphocytic choriomeningitis virus; measles virus; mumps virus; and rhinovirus
1A. Blue, WT (positive) control (104gene copies [gc]); green, H274Y (positive) control (104gc); orange, specificity test sample; red, no-template
control. Data from these plots is interpreted as described in the legend to Fig. 2.
3522 OPERARIO ET AL.J. CLIN. MICROBIOL.
methodology employed here has several advantages that would
make it attractive to hospital and public health laboratories.
MultiCode technology permits multiplexing as well as melt
curve analysis in real-time PCR, enabling testing of smaller
sample volumes, and a high level of discrimination between
specific and nonspecific amplification products. These ad-
vantages make isoC/isoG-based technology highly effective
at detecting specified targets and at quantifying mutant sub-
populations based on discrimination of a single nucleotide
polymorphism. As with all real-time PCR systems, this meth-
odology is best suited to laboratory facilities possessing real-
time equipment and having staff sufficiently trained and skilled
in molecular biology techniques. As such, this test system is not
appropriate for use in physician’s office laboratories.
For the detection of viral populations containing the H274Y
mutation and for discrimination of them from WT populations,
the assay has advantages over sequencing methods. Primers
and cycling conditions were optimized to achieve a limit of
detection of 100 gene copies per reaction, making the assay
usable on samples with viral loads too low to yield results by
either dideoxy sequencing or pyrosequencing. In samples con-
taining both WT and mutant neuraminidase gene targets, the
assay has the ability to detect as low as a 1% WT component
or a 0.1% H274Y component. This ability to reliably detect
mixed populations at low percentages is important in the
monitoring of patients for emergent drug resistance during
treatment. Due to its higher sensitivity compared to sequencing
methods, the assay presented here would be a preferable method
for detecting emergent drug resistance, as current pyrosequenc-
ing methods for the H274Y mutation are reported to only
reliably detect minor populations down to approximately a
10% component (7, 8). Specificity testing demonstrated the
ability to distinguish seasonal A/H1N1 from seasonal A/H3N2,
influenza B, and pandemic 2009 A/H1N1 viruses. While this
assay was designed to detect the H274Y oseltamivir-resistance
mutation, its level of specificity also makes the new assay useful
as an influenza subtyping test. Additionally, since the assay
requires no additional sample processing or manipulation after
the PCR step, results are obtained faster than dideoxy or
pyrosequencing. This assay is also more deployable than either
sequencing method, due to the ready availability of real-time
PCR instrumentation in clinical laboratory settings.
Testing of New York State influenza A/H1N1-positive pri-
mary samples from the 2007-2008 season confirmed the pres-
ence of the H274Y neuraminidase mutation in four samples
and only the WT genotype in all other samples from this
season. These real-time RT-PCR results matched all available
dideoxy sequencing data, lending validity to the data generated
by the new assay. The ability of the new assay to characterize
one additional sample as WT demonstrates its increased sen-
sitivity compared to that of dideoxy sequencing. The original
characterization of this specimen in 2008 indicated the speci-
men had an extremely low viral load. Previous attempts to
amplify this sample by conventional PCR for use in dideoxy
sequencing failed, producing no visible band in gel analysis.
Samples tested by the new assay from the 2005-2006 and 2006-
2007 seasons (including 24 samples not previously character-
ized by dideoxy sequencing) did not reveal the presence of
H274Y, in agreement with the documented emergence of this
mutation in the 2007-2008 influenza season. These data are
consistent with several possibilities: (i) only seasonal A/H1N1
viruses with WT neuraminidase were circulating in New York
State prior to the 2007-2008 season, (ii) the H274Y mutation
was present in specimens at a level lower than the 0.1% de-
tectable by the new assay, or (iii) the H274Y mutation was
present in seasonal A/H1N1 influenza prior to the 2007-2008
season but not at a frequency that would be detected without
a larger sample set than that available for this study.
Intensive and effective public health surveillance for drug
resistance in influenza is integral to the maintenance of neur-
aminidase inhibitors as useful treatment and prophylactic
agents. While oseltamivir is not currently effective against sea-
sonal A/H1N1, it remains effective against A/H3N2, influenza
B, and 2009 pandemic A/H1N1. The WHO has identified 285
cases of oseltamivir-resistant 2009 pandemic A/H1N1, with 64
cases identified by the CDC (6, 29). Viruses from these osel-
tamivir-resistant cases contain the H275Y neuraminidase mu-
tation, and approximately 8% of the global cases have had no
drug exposure or are suspected to have contracted resistant
virus by person-to-person transmission. Further intensified sur-
veillance efforts and improved detection methods for drug
resistance will be needed to ensure that resistance can be
identified in a timely manner and rapidly contained.
We thank Amy Dean, Sarah DuVall, Meghan Fuschino, Sara
Griesemer, Daryl Lamson, Jennifer Laplante, and Kim Rush-Wilson
at the Wadsworth Center and Scott Johnson at EraGen Biosciences for
technical assistance and helpful discussions, as well as Gino Battaglioli
at the Wadsworth Center for critical readings of the manuscript.
This work was supported in part by NIH grant T32 AI05542903A1,
CDC Cooperative Agreement no. U90/CCU216988, and CDC Coop-
erative Agreement no. U50/CCU223671.
This work is solely the responsibility of the authors and does not
necessarily represent the official views of the Centers for Disease
Control and Prevention.
1. Belshe, R. B., M. H. Smith, C. B. Hall, R. Betts, and A. J. Hay. 1988. Genetic
basis of resistance to rimantadine emerging during treatment of influenza
virus infection. J. Virol. 62:1508–1512.
TABLE 1. Comparative results between MultiCode-based testing
and dideoxy sequencing of seasonal A/H1N1 samples
from 2005 to 2008
Total no. of
No. (%) of samples
19b? 17 (97)
aSequencing data from Laplante et al. (14).
bNeuraminidase from these samples was not previously characterized by
cNA, not applicable.
VOL. 48, 2010H274Y MUTATION DETECTION USING isoC/isoG TECHNOLOGY 3523
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