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: email@example.com.
† Present address: Lucigen Corporation, 2120 W. Greenview Dr.,
Suite 9, Middleton, WI 53562.
?Published ahead of print on 28 July 2010.
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3524OPERARIO ET AL.J. CLIN. MICROBIOL.