JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2011, p. 3101–3102
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 8
A Synonymous Change in the Influenza A Virus Neuraminidase
Gene Interferes with PCR-Based Subtyping and Oseltamivir
Resistance Mutation Detection?
Rapid detection of the emergence of oseltamivir-resistant
influenza A virus is critically important for both public health
surveillance and the selection of appropriate antiviral therapy.
The most common oseltamivir-resistant influenza A (H1N1)
2009 virus genotype is a cytosine-to-thymine mutation at nu-
cleotide 823 of the neuraminidase (NA) gene (823C?T), re-
sulting in a histidine-to-tyrosine mutation at amino acid 275
(His275Tyr, N1 nomenclature). The close correlation of this
mutation with oseltamivir resistance has allowed the develop-
ment of numerous nucleic acid amplification-based methods
for its detection, including a duplex real-time, reverse trans-
criptase PCR (rRT-PCR) with allele-specific hydrolysis probes
that we implemented in our laboratory (Table 1). Here we
report the identification of a thymine-to-cytosine change at
nucleotide 822 (822T?C) that does not alter the amino acid
(Tyr274) but does interfere with probe binding in our labora-
tory-developed assay. Review of the literature suggests that
this variant may necessitate the redesign of many published
assays for influenza A (H1N1) 2009 virus oseltamivir resistance
Between 18 October 2010 and 12 April 2011, we tested 133
influenza A virus-positive respiratory specimens for the Tyr275
oseltamivir resistance mutation. Wild-type 2009 (H1N1) virus
NA RNA was detected in 71, while mutant RNA was found in
one, a sample from a pediatric heart transplant recipient on
oseltamivir. The remaining 61 samples did not give a signal in
the assay, but amplifiable influenza A virus matrix RNA was
confirmed using the CDC protocol. These specimens were
presumed to contain influenza A (H3N2) virus and were sent
to the Santa Clara County Public Health Laboratory or tested
in-house using commercial reagents (Gen-Probe Prodesse Pro-
Fast?) for type confirmation. H3 RNA was detected in 55,
while 2009 H1 RNA was unexpectedly detected in six.
After ruling out specimen mix-up and technical error, the
most likely explanation for these typing results was an issue
with our assay. Agarose gel electrophoresis of the rRT-PCR
mixtures demonstrated strong bands at the expected size (92
bp), indicating that the primers were functional and that se-
quences targeted by the probes were likely mismatched. Sanger
sequencing of the amplicons using the assay primers revealed
a single thymine-to-cytosine
(822T?C; Tyr274Tyr) in all six of the discrepant samples. Of
the 4,931 human 2009 (H1N1) virus neuraminidase sequences
in the NCBI database (accessed 30 May 2011), 32 contained
the same single nucleotide polymorphism (32/4,931; 0.65%).
These sequences were identified from viruses collected
throughout 2009 and 2010 in Portugal, Spain, Poland, Iran,
Kazakhstan, Canada, and the United States, suggesting the
random appearance and widespread distribution of this vari-
As this base change is directly adjacent to the mutation that
confers oseltamivir resistance, we evaluated the sequences of
primers and probes in published assays to determine whether
those assays might also be affected. We identified nine addi-
tional H275Y assays in the literature (1, 3–10) (Table 1). Of
these tests, only three took this variant into account (4, 6, 9).
Given that numerous strategies were employed to increase
target specificity, including minor groove binders, locked nu-
cleic acids, and RNA bases, the other assays would likely be
unable to tolerate this additional mismatch, and mutant or
wild-type sequence containing the variant may not be detected.
In addition, an erroneous subtype could be assigned if the
assay was not used in concert with subtyping based on another
region of the influenza A virus genome.
Introduction of a degenerate base in both probes in our
laboratory-developed assay (Table 1) allowed detection of
wild-type 2009 (H1N1) virus NA RNA in all six samples car-
rying this neuraminidase sequence variant. These observations
reinforce the importance of regular evaluation of assay target
sequences against newly available database sequences, partic-
ularly for RNA viruses like influenza A virus that have high
1. Bennett, S., R. N. Gunson, A. MacLean, R. Miller, and W. F. Carman.
2011. The validation of a real-time RT-PCR assay which detects influenza
A and types simultaneously for influenza A H1N1 (2009) and oseltamivir-
resistant (H275Y) influenza A H1N1 (2009). J. Virol. Methods 171:86–90.
2. Chen, N., B. A. Pinsky, B. P. Lee, M. Lin, and I. Schrijver. 2011. Ultrasen-
sitive detection of drug-resistant pandemic 2009 (H1N1) influenza A virus by
rare-variant-sensitive high-resolution melting-curve analysis. J. Clin. Micro-
3. Chidlow, G. R., et al. 2010. The detection of oseltamivir-resistant pandemic
influenza A/H1N1 2009 viruses using a real-time RT-PCR assay. J. Virol.
4. Hindiyeh, M., et al. 2010. Rapid detection of influenza A pandemic (H1N1)
2009 virus neuraminidase resistance mutation H275Y by real-time reverse
transcriptase PCR. J. Clin. Microbiol. 48:1884–1887.
5. Nukiwa, N., et al. 2010. Simplified screening method for detecting osel-
tamivir resistant pandemic influenza A (H1N1) 2009 virus by a RT-PCR/
restriction fragment length polymorphism assay. J. Virol. Methods 170:
6. Renaud, C., J. Kuypers, and L. Corey. 2010. Diagnostic accuracy of an
allele-specific reverse transcriptase-PCR assay targeting the H275Y oselta-
mivir resistant mutation in 2009 pandemic influenza A/H1N1 virus. J. Clin.
7. Suzuki, Y., et al. 2011. Identification of oseltamivir resistance among
pandemic and seasonal influenza A (H1N1) viruses by an His275Tyr
genotyping assay using the cycling probe method. J. Clin. Microbiol.
8. van der Vries, E., et al. 2010. Evaluation of a rapid molecular algorithm for
detection of pandemic influenza A (H1N1) 2009 virus and screening for a
key oseltamivir resistance (H275Y) substitution in neuraminidase. J. Clin.
9. Wang, B., et al. 2010. Detection of the rapid emergence of the H275Y
mutation associated with oseltamivir resistance in severe pandemic influenza
virus A/H1N1 09 infections. Antiviral Res. 87:16–21.
10. Wong, S., K. Pabbaraju, A. Wong, K. Fonseca, and S. J. Drews. 2011.
Development of a real-time RT-PCR assay for detection of resistance to
oseltamivir in influenza A pandemic (H1N1) 2009 virus using single nucleo-
tide polymorphism probes. J. Virol. Methods 173:259–265.
Benjamin A. Pinsky*
Stanford University Medical Center
*Phone: (650) 721-1896
Fax: (650) 736-1964
?Published ahead of print on 22 June 2011.
TABLE 1. Influenza A (H1N1) 2009 virus oseltamivir resistance assaysa
Assay and primer or
Sequence (5? to 3?)b
NA-WT probe V2
NA-tamifluR probe V2
SOIV NA 781F
SOIV NA 878R
SOIV Osel SEN
SOIV Osel RES
H1N1 swine 274Y RCA probe
H1N1 swine 274H RCA probe
RCA primer 1
RCA primer 2
aAbbreviations: rRT-PCR, real-time, reverse transcriptase PCR; RT-PCR/RFLP, reverse transcriptase PCR/restriction fragment length polymorphism; RT-PCR/
RCA, reverse transcriptase PCR/rolling circle amplification; FAM, 6-carboxyfluorescein; WT, wild type; BHQ, black hole quencher; NFQ, nonfluorescent quencher;
MGBNFQ, minor groove binder, nonfluorescent quencher; BBQ, blackberry quencher; ROX, 5-carboxy-X-rhodamine.
bBold underlining indicates position 823; bold italic indicates position 822.
cProbe uses locked nucleic acids; see reference 8 for details.
dProbe uses locked nucleic acids; see reference 1 for details.
eProbe uses an RNA base; see reference 7 for details.
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