5
Journal of Medical Virology 82:1224–1228 (2010) Antiviral Drug Susceptibilities of Seasonal Human Influenza Viruses in Lebanon, 2008–09 Season Hassan Zaraket, 1 * Reiko Saito, 1 Rima Wakim, 2 Carelle Tabet, 2 Fouad Medlej, 2 Mariam Reda, 2 Tatiana Baranovich, 1 Yasushi Suzuki, 1 Clyde Dapat, 1 Isolde Caperig-Dapat, 1 Ghassan S. Dbaibo, 1 and Hiroshi Suzuki 1 1 Division of Public Health, Department of Infectious Disease Control and International Medicine, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan 2 Division of Pediatric Infectious Diseases, Department of Pediatrics and Adolescent Medicine, American University of Beirut, Beirut, Lebanon The emergence of antiviral drug-resistant strains of the influenza virus in addition to the rapid spread of the recent pandemic A(H1N1) 2009 virus highlight the importance of surveillance of influenza in identifying new variants as they appear. In this study, genetic characteristics and antiviral susceptibility patterns of influenza sam- ples collected in Lebanon during the 2008–09 season were investigated. Forty influenza virus samples were isolated from 89 nasopharyngeal swabs obtained from patients with influenza-like illness. Of these samples, 33 (82.5%) were A(H3N2), 3 (7.5%) were A(H1N1), and 4 (10%) were B. All the H3N2 viruses were resistant to amantadine but were sensitive to oseltamivir and zanamivir; while all the H1N1 viruses were resistant to oseltamivir (possessed H275Y muta- tion, N1 numbering, in their NA) but were sensitive to amantadine and zanamivir. In the case of influenza B, both Victoria and Yamagata lineages were identified (three and one isolates each, respectively) and they showed decreased susceptibility to oseltamivir and zanamivir when compared to influenza A viruses. Influenza circulation patterns in Lebanon were very similar to those in Europe during the same season. Continued surveillance is important to fully elucidate influenza patterns in Lebanon and the Middle East in general, especially in light of the current influenza pandemic. J. Med. Virol. 82:1224–1228, 2010. ß 2010 Wiley-Liss, Inc. KEY WORDS: influenza; amantadine-resistant; oseltamivir-resistant; Lebanon; Middle East INTRODUCTION Influenza virus is the major cause of acute upper respiratory tract infections. Each year during the autumn and winter seasons, influenza A subtypes H3N2 and H1N1 and influenza B cause seasonal epidemics with significant health burdens [Stohr, 2002]. Vaccines are the mainstay for the prevention of influenza infections. However, the propensity of influenza viruses to change genetically and antigeni- cally elicits the need for continual modifications of vaccines strains [Carrat and Flahault, 2007; Zaraket et al., 2009b]. In addition to vaccines, antiviral drugs are available as important tools for mitigating influenza disease outcomes as well as prophylaxis. M2 channel- inhibitors; including amantadine and rimantadine, and neuraminidase (NA)-inhibitors; including oseltamivir and zanamivir, are the antivirals that are currently available against influenza [Memoli et al., 2008]. Never- theless, the decision on the proper usage antiviral medications has been complicated by the recent emer- gence of antiviral drug-resistant viruses [Hayden, 2006; Deyde et al., 2007; Reece, 2007; Meijer et al., 2009; Zaraket et al., 2009b]. Amantadine-resistant influenza A(H3N2) viruses emerged in 2003 in South-East Asia and are currently endemic worldwide. Amantadine- resistant influenza A(H1N1) emerged in 2005 and spread to most countries [Deyde et al., 2007; Hayden, 2006; Reece, 2007; Saito et al., 2007; Zaraket et al., Grant sponsor: Ministry of Foreign Affairs, Ministry of Health, Labor, and Welfare and Ministry of Education, Culture, Sports, Science and Technology (Japan). Hassan Zaraket’s present address is Present address: Division of Virology, Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, 38105 TN. E-mail: [email protected] *Correspondence to: Hassan Zaraket, Division of Public Health, Department of Infectious Disease Control and International Medicine, Niigata University, Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-Dori, Niigata City, Niigata Prefecture, 951-8510, Japan. E-mail: [email protected] Accepted 18 February 2010 DOI 10.1002/jmv.21795 Published online in Wiley InterScience (www.interscience.wiley.com) ß 2010 WILEY-LISS, INC.

Antiviral drug susceptibilities of seasonal human influenza viruses in Lebanon, 2008–09 season

Embed Size (px)

Citation preview

Journal of Medical Virology 82:1224–1228 (2010)

Antiviral Drug Susceptibilities of Seasonal HumanInfluenza Viruses in Lebanon, 2008–09 Season

Hassan Zaraket,1* Reiko Saito,1 Rima Wakim,2 Carelle Tabet,2 Fouad Medlej,2 Mariam Reda,2

Tatiana Baranovich,1 Yasushi Suzuki,1 Clyde Dapat,1 Isolde Caperig-Dapat,1

Ghassan S. Dbaibo,1 and Hiroshi Suzuki1

1Division of Public Health, Department of Infectious Disease Control and International Medicine, Niigata University,Graduate School of Medical and Dental Sciences, Niigata, Japan2Division of Pediatric Infectious Diseases, Department of Pediatrics and Adolescent Medicine,American University of Beirut, Beirut, Lebanon

The emergence of antiviral drug-resistant strainsof the influenza virus in addition to the rapidspread of the recent pandemic A(H1N1) 2009virus highlight the importance of surveillance ofinfluenza in identifying new variants as theyappear. In this study, genetic characteristics andantiviral susceptibility patterns of influenza sam-ples collected in Lebanon during the 2008–09season were investigated. Forty influenza virussamples were isolated from 89 nasopharyngealswabs obtained from patients with influenza-likeillness. Of these samples, 33 (82.5%) wereA(H3N2), 3 (7.5%) were A(H1N1), and 4 (10%)were B. All the H3N2 viruses were resistant toamantadine but were sensitive to oseltamivir andzanamivir; while all the H1N1 viruses wereresistant to oseltamivir (possessed H275Y muta-tion, N1 numbering, in their NA) but weresensitive to amantadine and zanamivir. In thecase of influenza B, both Victoria and Yamagatalineages were identified (three and one isolateseach, respectively) and they showed decreasedsusceptibility to oseltamivir and zanamivirwhen compared to influenza A viruses. Influenzacirculation patterns in Lebanon were very similarto those in Europe during the same season.Continued surveillance is important to fullyelucidate influenza patterns in Lebanon and theMiddle East in general, especially in light of thecurrent influenza pandemic. J. Med. Virol.82:1224–1228, 2010. � 2010 Wiley-Liss, Inc.

KEY WORDS: influenza; amantadine-resistant;oseltamivir-resistant; Lebanon;Middle East

INTRODUCTION

Influenza virus is the major cause of acute upperrespiratory tract infections. Each year during the

autumn and winter seasons, influenza A subtypesH3N2 and H1N1 and influenza B cause seasonalepidemics with significant health burdens [Stohr,2002]. Vaccines are the mainstay for the preventionof influenza infections. However, the propensity ofinfluenza viruses to change genetically and antigeni-cally elicits the need for continual modifications ofvaccines strains [Carrat and Flahault, 2007; Zaraketet al., 2009b]. In addition to vaccines, antiviral drugs areavailable as important tools for mitigating influenzadisease outcomes as well as prophylaxis. M2 channel-inhibitors; including amantadine and rimantadine, andneuraminidase (NA)-inhibitors; including oseltamivirand zanamivir, are the antivirals that are currentlyavailable against influenza [Memoli et al., 2008]. Never-theless, the decision on the proper usage antiviralmedications has been complicated by the recent emer-gence of antiviral drug-resistant viruses [Hayden, 2006;Deyde et al., 2007; Reece, 2007; Meijer et al., 2009;Zaraket et al., 2009b]. Amantadine-resistant influenzaA(H3N2) viruses emerged in 2003 in South-East Asiaand are currently endemic worldwide. Amantadine-resistant influenza A(H1N1) emerged in 2005 andspread to most countries [Deyde et al., 2007; Hayden,2006; Reece, 2007; Saito et al., 2007; Zaraket et al.,

Grant sponsor: Ministry of Foreign Affairs, Ministry of Health,Labor, and Welfare and Ministry of Education, Culture, Sports,Science and Technology (Japan).

Hassan Zaraket’s present address is Present address: Divisionof Virology, Department of Infectious Diseases, St. JudeChildren’s Research Hospital, Memphis, 38105 TN.E-mail: [email protected]

*Correspondence to: Hassan Zaraket, Division of Public Health,Department of Infectious Disease Control and InternationalMedicine, Niigata University, Graduate School of Medical andDental Sciences, 1-757, Asahimachi-Dori, Niigata City, NiigataPrefecture, 951-8510, Japan. E-mail: [email protected]

Accepted 18 February 2010

DOI 10.1002/jmv.21795

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2010 WILEY-LISS, INC.

2009b]. Moreover, in the 2007–08 season, oseltamivir-resistant A(H1N1) viruses prevailed in many Europeancountries and later spread to all the continents [Hurtet al., 2009; Meijer et al., 2009]. These factors haveraised concerns regarding the effectiveness of antiviralmedications and the usefulness of national antiviraldrug stockpiling programs.

In the 2008–09 season, reports have shown that mostof the influenza A(H3N2) viruses were resistant toamantadine, while most of the influenza A(H1N1)viruses were resistant to oseltamivir [WHO, 2008,2009a,b; CDC, 2009]. But regional variations in circu-lating types/subtypes as well as patterns of antiviraldrug resistance could also be observed. Influenzasurveillance in the Middle East is absent and data aboutcirculation and antiviral drug susceptibility patterns ofinfluenza are scarce. In the 2007–08 season, we startedstudying the influenza circulation in Lebanon, a MiddleEastern country, and found that influenza activityoccurs in the winter season [Zaraket et al., 2009a].Currently there is no active public influenza surveil-lance system running in Lebanon. This study aimed toinvestigate antiviral drug susceptibilities and geneticcharacteristics of influenza viruses isolated in Lebanonduring the 2008–09 season.

MATERIALS AND METHODS

Nasopharyngeal swabs were collected from patientswith influenza-like illness (fever and other respiratorysymptom such as cough and rhinorrhea) presenting atan outpatient clinic affiliated with the American Uni-versity of Beirut, located in the capital of Lebanon,Beirut. An informed consent was obtained from thepatients or their parents prior to sample collection.The study was approved by the University’s EthicsCommittee. Samples were stored in a virus transportmedium at �808C until being transported to NiigataUniversity’s Division of Public Health on dry ice forfurther analysis.

Swabs were inoculated on MDCK cells until thecharacteristic cytopathic effect could be observed. Onehundred microliters of virus culture supernatant wereused for RNA isolation using Extragen II1 (Kainos,Tokyo, Japan). First strand complementary DNA wasthen generated by reverse transcription using U11 andU12 primers [Hoffmann et al., 2001; Dapat et al., 2009].Typing and subtyping were performed by a real-timePCR method [CDC, 2007; Daum et al., 2007]. Twocycling probe real-time PCR assays were employed todetect single nucleotide polymorphisms at codonsencoding S31N in the M2 gene fragment [Suzuki et al.,

2009] and H275Y (N1 numbering) in the NA gene[Baranovich et al., 2009], which confers resistance toamantadine in H3N2 and resistance to oseltamivirin H1N1, respectively. Antiviral susceptibility wasalso tested by a phenotypic assay, tissue cultureinhibitory dose 50 (TCID50), for amantadine and afluorescent-based NA inhibition assay to determineinhibitory concentration 50 (IC50) for oseltamivirand zanamivir. Hemagglutinin genes of H3N2, H1N1,and B viruses were sequenced using specific primers[Dapat et al., 2009]. Generated sequences wereassembled and aligned using BioEdit 7.0.8.0 [Hall,1999] and phylogenetic relationships with referencestrains were inferred using MEGA (version 4.0) pro-gram [Tamura et al., 2007]. Sequences were deposited inthe Genbank database under accession numbers:CY047055–CY047070.

RESULTS

Between November 2008 to March 2009, 89 patientswith influenza-like symptoms participated in thestudy. The median age was 4.3 years, and 45 ofthe patients were male. Of the 89 cases, 40 (45%) werepositive for influenza by virus isolation; 33 caseswere influenza A(H3N2), 3 were influenza A(H1N1),and 4 were influenza B. Twenty-three patients of the89 (26%) had received the 2008–09 vaccine, out of whichseven had influenza A(H3N2) infection suggestingsuboptimal vaccination response to the H3N2strain (A/Brisbane/10/2007-like strain) included in the2008–09 vaccine. Twenty (50%) out of the 40 patientswith influenza infections reported household contactswith influenza-like illness.

In vitro testing of amantadine susceptibility (TCID50)revealed that all the H3N2 viruses were resistantto amantadine, but none of the H1N1 viruseswere resistant. On the other hand, NA inhibitionassay revealed that all the H1N1 viruses wereresistant to oseltamivir with the IC50 values rangingbetween 737 and 903 nM, while none of the H3N2viruses were resistant to oseltamivir (Table I).Genetically, the oseltamivir-resistant H1N1 virusespossessed a histidine-to-tyrosine substitution at posi-tion 275 (H275Y; N1 numbering) in the NA protein,known to confer oseltamivir resistance. All influenza Aviruses were sensitive to zanamivir, with the IC50sranging between 0.21 and 3.0 nM. The IC50s forinfluenza B viruses ranged between 38 and 61 nM foroseltamivir and between 19 and 81 nM for zanamivir(Table I). Notably, none of the 89 patients was onantiviral therapy prior to swab collection.

J. Med. Virol. DOI 10.1002/jmv

TABLE I. Susceptibility to Neuraminidase (NA) Inhibitors as Assessed by NA Inhibition Assays

Type/subtype n Oseltamivir IC50 (nM) Zanamivir IC50 (nM) Oseltamivir resistance (%)

A(H3N2) 33 1.26� 0.49 0.82� 0.65 0A(H1N1) 3 828.1� 84.1 1.73� 0.80 100B 4 50.1� 10.5 63� 29.4 0

Anti-viral Drug-Resistant Influenza in Lebanon 2008–09 1225

In HA phylogeny, all the H3N2 viruses clusteredtogether within a branch formed by isolates obtainedduring 2008–2009, and these were related to the WHOrecommended vaccine strain A/Brisbane/10/2007(Fig. 1). Seven of the 13 viruses sequenced possessedan I267V substitution in their HA. Strains isolated fromvaccinated patients did not show any characteristicmutations that might contribute to antigenic drift whencompared to the other viruses. The H1N1 virusesbelonged to the oseltamivir-resistant clade 2B andpossessed an A193T mutation (H3 numbering) thatwas characteristic of oseltamivir-resistant virusescirculating worldwide in the 2008–09 season (Fig. 1).Phylogenetic analysis of the HA of influenza B virusesrevealed that three of the four viruses belonged tothe Yamagata lineage and the remaining virus belongedto the Victoria lineage (Fig. 1). Interestingly, thisVictoria lineage virus was four times more susceptibleto zanamivir than the Yamagata lineage viruses(19.34 vs. 77.55), while no significant difference wasobserved in the IC50 of oseltamivir.

DISCUSSION

The emergence of antiviral drug resistance amongseasonal influenza viruses and the recent emergence ofthe pandemic (H1N1) 2009 virus have again reiterated

the importance of worldwide coverage of active surveil-lance of influenza virus infections. Data from MiddleEastern countries remains largely unavailable due tothe absence of public surveillance networks. In thisstudy, it was found that during the 2008–09 season inLebanon influenza A(H3N2) was dominant and theinfluenza A(H1N1) and influenza B viruses co-circu-lated. This pattern was similar to the influenza epidemicin Europe during the same season, where H3N2 virusesaccounted for 91% of identified viruses [WHO, 2009a].On the contrary, other regions in Asia (e.g., Japan andChina) and the US had a majority of H1N1 viruses[CDC, 2009].

All H3N2 viruses identified in the 2008–09 seasonin Lebanon were resistant to amantadine but sensitiveto NA-inhibitors, consistent with the pattern reportedworldwide [CDC, 2009; WHO, 2009a]. The amantadine-resistant H3N2 strain first emerged in 2003 in Chinaduring the avian H5N1 outbreaks [Bright et al., 2005].Since then, resistance rates have continued to increaseand now almost all H3N2 viruses maintain resistance toamantadine despite the limited usage of amantadineworldwide. On the other hand, all of the H1N1 virusesisolated in this study were sensitive to amantadine butresistant to oseltamivir. A high prevalence of oseltami-vir resistance was first reported in Europe in during the2007–08 season and spread gradually to most countries

J. Med. Virol. DOI 10.1002/jmv

Fig. 1. Phylogenetic relationships of HA genes from the influenza A(H3N2), the influenza A(H1N1), andthe influenza B viruses isolated in Lebanon during the 2008–09 season. Viruses isolated in this study areindicated in bold fonts. Reference vaccine strains are in black boxes. Bootstrap values are shown onbranches. Key amino acid changes relevant to the closest vaccine strain are indicated. H3 numbering wasused to indicate amino acid residues on both A(H3N2) and A(H1N1) phylogenies.

1226 Zaraket et al.

worldwide [Hauge et al., 2009; Meijer et al., 2009]. In the2008–09 season, oseltamivir resistance exceeded 90% inEuropean countries, Japan, South Korea, SouthAfrica [CDC, 2009; WHO, 2009a]. Moreover, variablerates of oseltamivir resistance were reported elsewhere(e.g., China, Malaysia, USA, Thailand) with very lowresistance rates being observed in countries such asTaiwan [Hurt et al., 2009], where most of H1N1 viruseswere oseltamivir-sensitive but resistant to amantadine.These data suggest the presence of regional variations inH1N1 circulation pattern.

In Lebanon, amantadine and oseltamivir were usedvery rarely in clinical practice until the advent of thepandemic (H1N1) influenza in 2009. This suggeststhat resistant-strains were introduced from externalsources, probably Europe, as seen by the similarcirculation patterns, due to the close proximityand frequent travel between Lebanon and Europe.Clinicians in Lebanon rarely use antiviral drugs againstinfluenza, but nevertheless, the availability of informa-tion on local antiviral susceptibility patterns is veryimportant for rational drug prescription.

Oseltamivir and zanamivir are also indicated fortreatment of influenza B. Previous reports indicateddecreased effectiveness of oseltamivir against influenzaB infections, but a controversy regarding the relation-ship between the age of the patient and the effectivenessof treatment remained [Sugaya et al., 2007; Kawai et al.,2008; Sato et al., 2008]. Zanamivir was shown to have agreater effect than oseltamivir for the treatment ofinfluenza B infections [Kawai et al., 2008]. However, theIC50 data indicates that both NA-inhibitors are equallyless effective against influenza B when compared toinfluenza A(H3N2). Interestingly, B/Lebanon/L06/2009of the Victoria lineage was four times more susceptible tozanamivir than the Yamagata lineage viruses in thisstudy. Sugaya et al. [2007] previously showed nosignificant differences in IC50s of NA-inhibitors onVictoria and Yamagata lineages. Nevertheless, thesetwo lineages continue to circulate and evolve independ-ently, warranting the need for continued investigationof their susceptibilities to NA-inhibitors. Although thenumber of strains in this study is limited and cannot beconsidered conclusive, it will be interesting to inves-tigate potential differences in NA-inhibitors’ activitieson the Yamagata and the Victoria lineages in the future.In the absence of clear cut-off values to judge viralstrains as resistant or susceptible, it remains essentialthat appropriate guidelines for antiviral susceptibilitytesting be made.

Until the emergence of amantadine-resistant H3N2 inthe 2003 season, it was widely thought that resistantinfluenza viruses did not have the potential for efficienttransmission and replication. However, continuedsuccessful circulation of the amantadine-resistant andthe oseltamivir-resistant viruses strongly suggest thatthese viruses could overcome previously believed fitnessdisadvantages. By acquiring certain mutations thatenable them to prevail [Simonsen et al., 2007]. InfluenzaA(H1N1) viruses that are resistant to both amantadine

and zanamivir were reported recently in Hong Kong[Cheng et al., 2009]. Despite these remain sporadiccases, it should not be surprising that these doubleresistant viruses become epidemic at anytime.

Amantadine-resistant viruses first emerged inAsia [Bright et al., 2005], while oseltamivir-resistantviruses emerged in Europe [Hauge et al., 2009], and thepandemic (H1N1) 2009 viruses first appeared in Mexico[Smith et al., 2009] suggesting that new variants ofinfluenza viruses could emerge anywhere and havethe potential to spread worldwide. Altogether, theseobservations emphasize the importance of surveillanceprograms that cover most countries in the world forearly detection of variant strains.

ACKNOWLEDGMENTS

We are very thankful Akime Watanabe for herexcellent technical assistance, Y. Kato for intensivesecretarial work, and Lindsey Gibbon for editing themanuscript.

REFERENCES

Baranovich T, Saito R, Suzuki Y, Zaraket H, Dapat C, Caperig-Dapat I,Oguma T, Shabana II, Saito T, Suzuki H. 2010. Emergence ofH274Y oseltamivir-resistant A(H1N1) influenza viruses in Japanduring the2008–2009 season. J Clin Virol 47:23–28.

Bright RA, Medina MJ, Xu X, Perez-Oronoz G, Wallis TR, Davis XM,Povinelli L, Cox NJ, Klimov AI. 2005. Incidence of adamantaneresistance among influenza A (H3N2) viruses isolated worldwidefrom 1994 to 2005: A cause for concern. Lancet 366:1175–1181.

Carrat F, Flahault A. 2007. Influenza vaccine: The challenge ofantigenic drift. Vaccine 25:6852–6862.

Centers for Disease Control and Prevention. Realtime PCR protocol fordetection and characterization of influenza. Version 2007. Atlanta:Centers for Disease Control and Prevention.

Centers for Disease Control and Prevention. VRPBAC. http://www.fda.gov/ohrms/dockets/ac/09/briefing/2009-4416B1-1.pdf(accessed May 05, 2009).

Cheng PK, Leung TW, Ho EC, Leung PC, Ng AY, Lai MY, Lim WW.2009. Oseltamivir- and amantadine-resistant influenza viruses A(H1N1). Emerg Infect Dis 15:966–968.

Dapat C, Saito R, Kyaw Y, Naito M, Hasegawa G, Suzuki Y, Dapat IC,Zaraket H, Cho TM, Li D, Oguma T, Baranovich T, Suzuki H. 2009.Epidemiology of human influenza A and B viruses in Myanmar from2005 to 2007. Intervirology 52:310–320.

Daum LT, Canas LC, Arulanandam BP, Niemeyer D, Valdes JJ,Chambers JP. 2007. Real-time RT-PCR assays for type and subtypedetection of influenza A and B viruses. Influenza Other Respir Virus1:167–175.

Deyde VM, Xu X, Bright RA, Shaw M, Smith CB, Zhang Y, Shu Y,Gubareva LV, Cox NJ, Klimov AI. 2007. Surveillance of resistanceto adamantanes among influenza A(H3N2) and A(H1N1) virusesisolated worldwide. J Infect Dis 196:249–257.

Hall TA. 1999. BioEdit: A user-friendly biological sequence alignmenteditor and analysis program for Windows 95/98/NT. Nucl AcidsSymp 41:95–98.

Hauge SH, Dudman S, Borgen K, Lackenby A, Hungnes O. 2009.Oseltamivir-resistant influenza viruses A (H1N1), Norway,2007–08. Emerg Infect Dis 15:155–162.

Hayden FG. 2006. Antiviral resistance in influenza viruses—Implica-tions for management and pandemic response. N Engl J Med 354:785–788.

Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. 2001. Universalprimer set for the full-length amplification of all influenza Aviruses. Arch Virol 146:2275–2289.

Hurt AC, Ernest J, Deng YM, Iannello P, Besselaar TG, Birch C, BuchyP, Chittaganpitch M, Chiu SC, Dwyer D, Guigon A, Harrower B, KeiIP, Kok T, Lin C, McPhie K, Mohd A, Olveda R, Panayotou T,Rawlinson W, Scott L, Smith D, D’Souza H, Komadina N, Shaw R,

J. Med. Virol. DOI 10.1002/jmv

Anti-viral Drug-Resistant Influenza in Lebanon 2008–09 1227

Kelso A, Barr IG. 2009. Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asiaand South Africa. Antiviral Res 83:90–93.

Kawai N, Ikematsu H, Iwaki N, Maeda T, Kanazawa H, Kawashima T,Tanaka O, Yamauchi S, Kawamura K, Nagai T, Horii S, Hirotsu N,Kashiwagi S. 2008. A comparison of the effectiveness of zanamivirand oseltamivir for the treatment of influenza A and B. J Infect 56:51–57.

Meijer A, Lackenby A, Hungnes O, Lina B, van-der-Werf S, SchweigerB, Opp M, Paget J, van-de-Kassteele J, Hay A, Zambon M. 2009.Oseltamivir-resistant influenza virus A (H1N1), Europe, 2007–08season. Emerg Infect Dis 15:552–560.

Memoli MJ, Morens DM, Taubenberger JK. 2008. Pandemic andseasonal influenza: Therapeutic challenges. Drug Discov Today 13:590–595.

Reece PA. 2007. Neuraminidase inhibitor resistance in influenzaviruses. J Med Virol 79:1577–1586.

Saito R, Li D, Suzuki Y, Sato I, Masaki H, Nishimura H, Kawashima T,Shirahige Y, Shimomura C, Asoh N, Degawa S, Ishikawa H, Sato M,Shobugawa Y, Suzuki H. 2007. High prevalence of amantadine-resistance influenza a (H3N2) in six prefectures, Japan, in the2005–2006 season. J Med Virol 79:1569–1576.

Sato M, Saito R, Sato I, Tanabe N, Shobugawa Y, Sasaki A, Li D, SuzukiY, Sato M, Sakai T, Oguma T, Tsukada H, Gejyo F, Suzuki H. 2008.Effectiveness of oseltamivir treatment among children withinfluenza A or B virus infections during four successive winters inNiigata City, Japan. Tohoku J Exp Med 214:113–120.

Simonsen L, Viboud C, Grenfell BT, Dushoff J, Jennings L, Smit M,Macken C, Hata M, Gog J, Miller MA, Holmes EC. 2007. Thegenesis and spread of reassortment human influenza A/H3N2viruses conferring adamantane resistance. Mol Biol Evol 24:1811–1820.

Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG,Ma SK, Cheung CL, Raghwani J, Bhatt S, Peiris JS, Guan Y,

Rambaut A. 2009. Origins and evolutionary genomics of the 2009swine-origin H1N1 influenza A epidemic. Nature 459:1122–1125.

Stohr K. 2002. Influenza—WHO cares. Lancet Infect Dis 2:517.

Sugaya N, Mitamura K, Yamazaki M, Tamura D, Ichikawa M, KimuraK, Kawakami C, Kiso M, Ito M, Hatakeyama S, Kawaoka Y. 2007.Lower clinical effectiveness of oseltamivir against influenza Bcontrasted with influenza A infection in children. Clin Infect Dis44:197–202.

Suzuki Y, Saito R, Zaraket H, Dapat C, Caperig-Dapat I, Suzuki H.2009. Rapid and specific detection of amantadine-resistant Ser31-Asn mutated influenza A viruses by cycling probe method. J ClinMicrobiol 43:57–63.

Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecularevolutionary genetics analysis (MEGA) software version 4.0. MolBiol Evol 24:1596–1599.

World HealthOrganization.2008. InfluenzaA(H1N1) virus resistance tooseltamivir—2008 influenza season, southern hemisphere. http://www.who.int/csr/disease/influenza/H1N1webupdate20082008_kf.pdf (accessed December 28, 2008).

World Health Organization Influenza Center London. 2009a. February2009 Report. http://www.nimr.mrc.ac.uk/wic/report/docs/interim_report_feb_2009.pdf (accessed May 05, 2009).

World Health Organization. 2009b. Influenza A(H1N1) virus resis-tance to oseltamivir—2008/2009 influenza season, northern hemi-sphere. http://www.who.int/csr/disease/influenza/H1N1webupdate20090318%20ed_ns.pdf (accessed May 05, 2009).

Zaraket H, Dbaibo G, Salam O, Saito R, Suzuki H. 2009a. Influenzavirus infections in Lebanese children in the2007–2008 season. JpnJ Infect Dis 62:137–138.

Zaraket H, Saito R, Sato I, Suzuki Y, Li D, Dapat C, Caperig-Dapat I,Oguma T, Sasaki A, Suzuki H. 2009b. Molecular evolution of humaninfluenza A viruses in a local area during eight influenza epidemicsfrom 2000 to 2007. Arch Virol 154:285–295.

J. Med. Virol. DOI 10.1002/jmv

1228 Zaraket et al.