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Journal of Clinical Microbiology, May 2009, p. 1424-1427, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02396-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Oseltamivir-Resistant Influenza A Viruses Circulating in Japan

Daisuke Tamura,^1* Keiko Mitamura,² Masahiko Yamazaki,³ Motoko Fujino,⁴ Mari Nirasawa,⁴ Kazuhiro Kimura,⁵ Maki Kiso,¹ Hideaki Shimizu,⁶ Chiharu Kawakami,⁷ Satoshi Hiroi,⁸ Kazuro Takahashi,⁸ Mami Hata,⁹ Hiroko Minagawa,⁹ Yoshiaki Kimura,¹⁰ Satoko Kaneda,¹⁰ Shigeo Sugita,¹¹ Taisuke Horimoto,¹ Norio Sugaya,¹² and Yoshihiro Kawaoka^1,13,14

Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan,¹ Department of Pediatrics, Eijyu General Hospital, Tokyo, Japan,² Department of Pediatrics, Zama Children's Clinic, Kanagawa, Japan,³ Department of Pediatrics, Saiseikai Central Hospital, Tokyo, Japan,⁴ Department of Pediatrics, Isehara Kyodo Hospital, Kanagawa, Japan,⁵ Kawasaki City Institute of Public Health, Kanagawa, Japan,⁶ Yokohama City Institute of Public Health, Kanagawa, Japan,⁷ Osaka Prefectural Institute of Public Health, Osaka, Japan,⁸ Aichi Prefectural Institute of Public Health, Aichi, Japan,⁹ Tottori Prefectural Institute of Public Health and Environmental Science, Tottori, Japan,¹⁰ Equine Research Institute, Japan Racing Association, Tochigi, Japan,¹¹ Department of Pediatrics, Keiyu Hospital, Kanagawa, Japan,¹² International Research Center for Infectious Disease, Institute of Medical Science, University of Tokyo, Tokyo, Japan,¹³ Department of Pathobiological Science, School of Veterinary Medicine, University of Wisconsin—Madison, Madison, Wisconsin,¹⁴

Received 14 December 2008/ Returned for modification 27 January 2009/ Accepted 17 February 2009

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Surveillance studies of the influenza viruses circulating in Europe and other countries in 2007 and 2008 have revealed rates of resistance to oseltamivir of up to 67% among H1N1 viruses. In the present study, we examined 202 clinical samples obtained from patients infected with H1N1 virus in Japan in 2007 and 2008 for oseltamivir resistance and found that three were oseltamivir resistant (1.5%). The 50% inhibitory concentrations (IC₅₀s), as measured by a sialidase inhibition assay with these drug-resistant viruses, were >100-fold higher than those of the nonresistant viruses (median IC₅₀, 12.6 nmol/liter). The His274Tyr (strain N2 numbering) mutation of the neuraminidase protein, which is known to confer oseltamivir resistance, was detected in these three isolates. Phylogenetic analysis showed that one virus belonged to a lineage that is composed of drug-resistant viruses isolated in Europe and North America and that the other two viruses independently emerged in Japan. Continued surveillance studies are necessary to observe whether these viruses will persist.

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Seasonal influenza is an important disease, the epidemiology of which is characterized by annual epidemics that occur throughout the world every year. It remains a leading cause of death and is responsible for substantial economic losses worldwide (1). Furthermore, avian influenza virus and other novel influenza virus strains have the potential to cause pandemics. In addition to vaccines, two classes of drugs are important for the control of influenza: M2 ion channel inhibitors (amantadine and rimantadine) and neuraminidase (NA) inhibitors (oseltamivir and zanamivir). The widespread use of antiviral drugs, however, has the potential to promote the emergence of drug-resistant viruses (4, 6, 14). In fact, the majority of H3N2 and H1N1 viruses in humans are now amantadine and rimantadine resistant (11, 12).

Influenza virus surveillance studies during the Northern Hemisphere's winter seasons prior to the 2007-2008 season revealed no oseltamivir-resistant H1N1 virus isolates in Europe or Japan and a less than 1% prevalence in the United States (2, 7, 15), indicating the limited transmission of such resistant viruses in these areas. However, there has recently been an increase in the prevalence of oseltamivir-resistant H1N1 viruses. These viruses were shown to harbor a specific mutation in the NA protein in which tyrosine is substituted for histidine at amino acid position 274. Several countries in Europe and North America have recently reported an increased prevalence of oseltamivir-resistant H1N1 viruses (13, 16). To date, oseltamivir-resistant H1N1 viruses have been detected in 26 countries around the world. In particular, 681 of 2,740 (25%) isolates in Europe and 210 of 1,342 (16%) isolates in North America (13, 16) were found to carry the His274Tyr mutation in the NA protein, which is known to confer a high level of resistance to oseltamivir. When viruses bearing this mutation were tested in a sialidase inhibition assay, they demonstrated a 400-fold reduction in susceptibility to oseltamivir when the 50% inhibitory concentrations (IC₅₀s), or the concentrations of oseltamivir required to inhibit 50% of the sialidase activity of the virus, of the viruses with and without the mutation were compared (7).

Oseltamivir is widely used in Japan. In fact, more than 70% of all oseltamivir doses are administered in Japan. More specifically, 7 million, 5.3 million, and 8.5 million doses of oseltamivir were used in the 2002-2003, 2003-2004, and 2004-2005 influenza seasons, respectively (10). The World Health Organization recently presented data that indicate that the emerging rate of oseltamivir-resistant H1N1 virus is approximately 2% in Japan (16). To understand the origin of oseltamivir-resistant H1N1 viruses isolated in Japan and their genetic relationship to the oseltamivir-resistant H1N1 viruses isolated in other countries, we examined H1N1 viruses isolated in Japan in 2007-2008, determined their oseltamivir IC₅₀s, and performed phylogenetic analysis of their viral NA and hemagglutinin (HA) genes.

(The information described in this article has been included in a patent application for novel amino acid substitutions and methods for detecting them.)

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Clinical samples. We collected pharyngeal or nasal swab specimens from patients with influenza who visited the pediatric services at five hospitals and who were diagnosed with influenza virus infection by use of a rapid influenza diagnosis kit. A total of 152 H1N1 influenza viruses obtained from the patients in these five hospitals were used in this study. In addition, 50 H1N1 isolates were obtained from three institutes of public health: the Osaka Prefecture Institute of Public Health, the Aichi Prefectural Institute of Public Health, and the Tottori Prefecture Institute of Public Health and Environmental Science. Thus, we tested a total of 202 influenza H1N1 virus isolates that were isolated from samples collected between November 2007 and April 2008.

Oral informed consent was obtained from the parents or legal guardians of the 152 patients enrolled in the study, and the study was conducted with the approval of the ethics committees of four of the hospitals (in the case of the one hospital without an ethics committee, the activities of the study were undertaken under the auspices of informed consent). The 50 samples obtained from the three institutes of public health were originally obtained for influenza virus surveillance and were collected at hospitals. Oral informed consent was obtained from all patients. The study with patient samples was conducted with the approval of the ethics committee of the Institute of Medical Science, University of Tokyo (approval number 20-35-0930).

Sialidase sensitivity to oseltamivir. For the in vitro studies, we used oseltamivir carboxylate, the active metabolite of the ethyl ester prodrug oseltamivir phosphate. Madin-Darby canine kidney cells were used for virus isolation and to determine the viral subtype by conventional HA and NA inhibition assays. To assess the sensitivities of the H1N1 viruses to oseltamivir, we determined the IC₅₀s of these viruses in a sialidase activity assay with 2'-(4-methylumbelliferyl)--D-N-acetylneuraminic acid (MUNANA; Sigma, St. Louis, MO) as the substrate at a final concentration of 0.1 mmol/liter. Ten microliters of a virus dilution (predetermined to contain sialidase activity in the range of 800 to 1,200 fluorescence units in this assay) and 10 µl of oseltamivir (0.01 nmol/liter to 10 µmol/liter) in calcium-2-(N-morpholino)ethanesulfonic acid [33 mmol 2-(N-morpholino)ethanesulfonic acid, 4 mmol/liter CaCl₂, pH 6.0] were mixed, and the mixture was incubated at 37°C for 30 min. Following incubation, 30 µl of 0.1 mmol/liter MUNANA was added to the mixtures. The mixtures were further incubated at 37°C for 60 min. The reaction was stopped by the addition of 150 µl of 0.1 mmol/liter sodium hydroxide in 80% ethanol (pH 10.0). We quantified the fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The results are reported as the means of duplicate IC₅₀s.

Sequence analyses. Viral RNA was extracted from viruses by use of a QIAamp viral RNA minikit (Qiagen, Hilden, Germany). For the reverse transcription reactions, we used influenza A virus universal primer 5'-AGCAAAAGCAGG-3', which is complementary to the 3' end of virus RNA, and reverse transcriptase (Superscript III; Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. The cDNA products were then used as the template for the amplification of the NA and HA genes by a standard PCR method (with Phusion high-fidelity DNA polymerase; Finnzymes, Espoo, Finland). The PCR products were separated on a 1% agarose gel. These PCR products were submitted to DNA sequencing to determine the complete sequences of the NA genes by cycle. Briefly, DNA sequencing was performed by the use of a BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) on an Applied Biosystems 3100 or 3130Xl automatic sequencer with NA-specific primers, according to the manufacturer's instructions. The HA genes of the isolates with reduced susceptibility to oseltamivir were similarly sequenced by using HA-specific primers.

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H1N1 viruses with reduced sensitivity to oseltamivir. To ascertain the prevalence of oseltamivir resistance among H1N1 viruses that were isolated from patients prior to oseltamivir treatment in Japan, we determined the oseltamivir carboxylate IC₅₀s of 202 viruses isolated in five prefectures in Japan. The IC₅₀s of the following three viruses were substantially higher than those of the other viruses: A/Yokohama/UTZS-48/08, 1,184.9 nmol/liter; A/Yokohama/UTKEI-62/08, 1,847.5 nmol/liter; and A/Tottori/UT-52/08, 2,880 nmol/liter. The median IC₅₀ for the other 199 isolates was 12.6 nmol/liter (range, 1.46 to 61.3 nmol/liter) (Fig. 1). Thus, the prevalence of oseltamivir-resistant viruses in our study was 1.5% (3/202 viruses).

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FIG. 1. Distribution of IC50s of oseltamivir carboxylate for influenza A (H1N1) viruses isolated from 202 patients. The inhibitory effect of oseltamivir carboxylate on the sialidase activity of the influenza virus was assessed by a fluorimetric assay. The box indicates the interquartile range, the vertical blue dotted line indicates the mean value, and the bars to the left and right of the box indicate 1.5 times the interquartile range. The oseltamivir IC50s for the three strains isolated in Japan, A/Yokohama/UTZS-48/08, A/Yokohama/UTKEI-62/08, and A/Tottori/UT-52/08, were 1,184.9 nmol/liter, 1,847.5 nmol/liter, and 2,880 nmol/liter, respectively. The median IC50 of the other 199 strains was 12.6 nmol/liter (range, 1.46 to 61.3 nmol/liter).

Sequence analysis of oseltamivir-resistant isolates. To understand the molecular basis of the reduced sensitivity to oseltamivir that was observed, we analyzed the NA and HA genes from the viruses exhibiting this property. Our sequence analysis revealed an amino acid substitution, His274Tyr, in the NA proteins of the three isolates that exhibited reduced oseltamivir sensitivity. The His274Tyr mutation in the NA protein is located in the sialidase active site (3). No other difference known to confer oseltamivir resistance was observed in the amino acid sequences of these NA proteins. In addition, the HA proteins of the three resistant virus isolates in this study had no specific amino acid changes that may alter receptor binding properties.

To understand the genetic relationship of these three oseltamivir-resistant viruses to one another and to those that were prevalent in Europe and North America, the NA genes of the three oseltamivir-resistant viruses that were identified were phylogenetically analyzed together with the NA genes of other viruses (8). The analysis showed that the NA genes of oseltamivir-resistant viruses A/Yokohama/UTZS-48/08 (the sample from which this virus was isolated was collected on 5 February 2008) and A/Yokohama/UTKEI-62/08 (the sample from which this virus was isolated was collected on 15 February 2008), which differed by only 3 nucleotides, were highly related to one another (Fig. 2) and belonged to the same cluster, cluster A. The other two viruses in this cluster, A/Tokyo/UTZS-14/08 and A/Yokohama/UTZS-10/08, were isolated in Japan and were found to be oseltamivir sensitive. In contrast, A/Tottori/UT-52/08 (the sample from which this virus was isolated was collected on 29 February 2008) belonged to cluster B, which is composed of oseltamivir-resistant viruses isolated in Europe and the United States. In fact, the NA genes of A/Tottori/UT-52/08 and A/England/557/07 differed by only 5 nucleotides, leading to a single amino acid difference. Thus, our results suggest the independent generation in Japan of the oseltamivir-resistant H1N1 viruses A/Yokohama/UTZS-48/08 and A/Yokohama/UTKEI-62/08 belonging to cluster A and the transmission of oseltamivir-resistant virus A/Tottori/UT-52/08, which was likely derived from a virus belonging to cluster B. Whether the oseltamivir-resistant viruses in cluster A will become widespread is not known. However, those in cluster B appear to have acquired this ability, as they have been isolated in Europe, the United States, and Japan.

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FIG. 2. Phylogenetic tree of NA genes of H1N1 viruses isolated over a 17-year period (1991 to 2008). Strains labeled in red are oseltamivir resistant and possess the H274Y mutation in the NA protein. The oseltamivir-resistant viruses isolated in this study are boxed. The phylogenetic tree was derived from the nucleotide sequences of 888 NA genes by use of the neighbor-joining method. A similar result was obtained by use of the maximum-parsimony method (data not shown).

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Unexpectedly, the incidence of oseltamivir-resistant H1N1 viruses increased globally in the 2007-2008 influenza season. Since the rate of spontaneous mutation of influenza viruses is about 1 in 10,000 (9, 17) and a single mutation from His to Tyr at position 274 (strain N2 numbering) confers oseltamivir resistance to viruses, mutant viruses are expected to be present when more than 10,000 infectious virus particles are present. However, a virus with a His274Tyr mutation in NA that exists with a prevalence of 1 in 10,000 would not become dominant in patients unless the environment favors the virus with the His274Tyr mutation, i.e., in the case of treatment with oseltamivir. Thus, the oseltamivir-resistant virus must have become dominant in oseltamivir-treated patients. What is remarkable is that the oseltamivir-resistant virus with the His274Tyr mutation has been shown to grow less efficiently than wild-type virus in ferrets (5), suggesting that the resistant virus could not effectively compete with wild-type oseltamivir-sensitive virus in an environment where oseltamivir is not present. The fact that the oseltamivir-resistant virus has been transmitted among humans suggests that the resistant virus must have acquired additional mutations that would make the virus competitive against wild-type viruses. Additional studies are needed to identify such mutations to further our understanding not only of the mechanisms by which drug-resistant viruses become prevalent but also of how viruses evolve in general.

It is interesting that the transmission of oseltamivir-resistant H1N1 viruses in humans was first recognized in Europe and North America and that the prevalence of such viruses reported in Norway was substantially higher than that reported in other countries, especially considering that the rate of use of oseltamivir in these countries is not as high as that in Japan. However, even though the amounts of oseltamivir used in these countries are not as large as those used in Japan, the drug is still used. Thus, it is possible that the oseltamivir-resistant H1N1 viruses currently in circulation among humans emerged in Europe, as resistant viruses have been isolated at high rates in that area.

The oseltamivir-resistant H1N1 viruses isolated in Japan belong to two distinct lineages (Fig. 2). A/Tottori/UT-52/08 was closely related to viruses isolated in Europe and North America, whereas A/Yokohama/UTZS-48/08 and A/Yokohama/UTKEI-62/08 belong to a lineage that is distinct from that to which A/Tottori/UT-52/08 belongs. Thus, A/Tottori/UT-52/08 might have been imported into Japan and A/Yokohama/UTZS-48/08 and A/Yokohama/UTKEI-62/08 may have emerged independently in Japan. It is currently not known whether the last two viruses will become transmissible.

Amantadine- and rimantadine-resistant influenza viruses have been isolated from humans since the clinical use of these drugs was initiated. However, resistant viruses did not become prevalent until recently. We found the transmission of oseltamivir-resistant influenza B viruses in Japan during the 2006-2007 influenza season (4), but whether these viruses will continue to spread among humans is unknown. Whether the oseltamivir-resistant H1N1 viruses that were transmitted during that season will continue to spread among humans in subsequent seasons and whether oseltamivir-resistant H3N2 viruses will emerge and become widely transmissible also remain unknown. Such scenarios would require a substantial change in treatment strategies. Thus, close monitoring for oseltamivir-resistant influenza viruses is essential.

 
This work was supported by a grant-in-aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by research grants from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service.

Y. Kawaoka has received speaker's honoraria from Chugai Pharmaceuticals, Novartis, Sankyo, Toyama Chemical, Wyeth, and GlaxoSmithKline and grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, and Toyama Chemical and is a founder of FluGen. N. Sugaya has received a travel grant from Roche to attend a meeting on avian influenza virus, has received a speaker's honorarium from Daiichi Sankyo, and is currently conducting studies evaluating compounds from Daiichi Sankyo. K. Mitamura has received a speaker's honorarium from GlaxoSmithKline. We have no other disclosures to report.

 
* Corresponding author. Mailing address: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5310. Fax: 81-3-5449-5408. E-mail: kawaoka{at}ims.u-tokyo.ac.jp

Published ahead of print on 4 March 2009.

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Journal of Clinical Microbiology, May 2009, p. 1424-1427, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.02396-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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