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Objective: In the beginning of the 2007-2008 influenza season in Northern Hemisphere, the frequency of influenza A (H1N1) viruses bearing a previously defined oseltamivir resistance increased dramatically. This is conferring to the amino acid exchange from histidine to tyrosine at position 274 (H274Y) in neuraminidase (NA) gene. The overall frequency of oseltamivir resistance in influenza A (H1N1) strains in Europe was reported as being 25%. Although it varied between countries, it was shown that the highest percentage was in Norway (67%), and the lowest was in Spain (2%). In this study, it was aimed to evaluate the oseltamivir resistance in influenza A H1N1 isolates from Turkey during the 20072008 influenza season. Method: During November 2007 – May 2008, 20 samples were randomly selected between 73 influenza A (H1N1) positive samples detected with real-time RTPCR in National Influenza Center (NIC) of Refik Saydam National Public Health Agency (RSNPHA). To detect such resistant viruses in Turkey, an RT-PCR assay was performed targeting amino acid position at 274 in NA gene of H1N1 influenza strain to investigate the presence or absence of histidine to tyrosine mutation. OZET Amac: Kuzey yarim kurede 2007–2008 influenza sezonunun baslangicindan itibaren dahaonce tanimlanmis olan influenza A (H1N1) virusunun oseltamivir direncinin arttigisaptanmistir. Bu direncin gostergesi, noraminidaz (NA) geninin 274. aminoasidinin (H274Y) histidinden tirozine donusumu ile ortaya konmaktadir. Avrupa’da influenza A (H1N1) suslari arasinda oseltamivire direnc orani %25 olarak saptanmistir. Bu oran ulkeler arasinda farklilik gostermekle birlikte Norvec’te en yuksek (%67), Ispanya’da en dusuk (%2) olarak tespit edilmistir. Bu calismada 2007-2008 influenza sezonunda Turkiye’deki influenza A H1N1 izolatlarinda oseltamivir direncini tespit etmek amaclanmistir. Yontem: Refik Saydam Hifzissihha Merkezi Baskanligi (RSHMB) Ulusal Influenza Merkezi’nde 2007 Kasim 2008 Mayis aylari arasinda real-time RT-PCR ile test edilerek influenza A (H1N1) pozitif olarak bulunan toplam 73 ornek arasindan rastlantisal olarak 20 ornek secilmistir. Oseltamivir direncini tespit etmek icin influenza A H1N1 suslarinin NA gen bolgesi 274. aminoasitini hedefleyen RT-PCR islemi, histidinden tirozine donusumu gostermek Gelis Tarihi / Received : Kabul Tarihi / Accepted : Iletisim / Corresponding Author : Nurhan ALBAYRAK RSHMB, Salgin Hast. Ars., Mud. Vir. Ref. ve Ars. Lab., Ulusal Inf. Merk., Sihhiye, ANKARA Tel : +90 312 458 22 11 E-posta / E-mail : nurhanalbayrak@hotmail.com 25.04.2011 15.02.2012 Turk Hijyen ve Deneysel Biyoloji Dergisi DOI ID : 10.5505/TurkHijyen.2012.23600 Turkiye’de 2007 – 2008 influenza sezonunda oseltamivir direncli influenza A (H1N1) viruslerinin H274Y mutasyonu ile saptanmasi Carhan A, Albayrak N, Altas AB, Uyar Y, Ozkaya E. Turkiye’de 2007 – 2008 influenza sezonunda oseltamivir direncli influenza A (H1N1) viruslerinin H274Y mutasyonu ile saptanmasi. Turk Hij Den Biyol Derg, 2012; 69(1): 1-6. Cilt 69  Sayi 1  2012 2 The prevalence of oseltamivir-resistant influenza viruses A (H1N1) dramatically increased worldwide at the beginning of 2007-2008 influenza season in the northern hemisphere. Recent reports indicated that by early 2009 most influenza A (H1N1) strains were resistant to oseltamivir (a neuraminidase inhibitor). These were associated with a specific “histidine-totyrosine mutation” at position 274 (H274Y; H275Y in N1 numbering system) in the neurominidase (NA) protein that confers high-level resistance to oseltamivir (15). An increased number of influenza A viruses (H1N1) with resistance to oseltamivir was first reported by World Health Organization (WHO, Norway) in January 2008 and high levels of oseltamivir resistance were determined among influenza A (H1N1) viruses in European countries and USA (4,6-8). WHO Global Influenza Surveillance Network (GISN) showed that 16% of community isolates (0%–67% by country) of influenza A viruses (H1N1) were oseltamivir resistant in the 2007 – 2008 season. The overall frequency of oseltamivir resistance in influenza A (H1N1) strains from Europe was approximately 25%. The frequency of seasonal influenza A (H1N1) viruses resistance to oseltamivir varied between countries. The highest percentage was observed in Norway (67%), followed by France (47%), while increased levels of resistance to oseltamivir were detected worldwide (2-6,8). The aim of this study was to determine the resistance to oseltamivir (neuraminidase inhibitor) in some influenza A H1 isolates obtained during the 2007-2008 influenza season in central and eastern parts of Turkey. In order to detect such histidine to tyrosine mutation in amino acid position 274 in NA gene, RT-PCR assay was performed. INTRODUCTION INFLUENZA A (H1N1) OSELTAMIVIR DIRENCI icin uygulanmistir. Influenza viruslerinin NA geni sekanslanmis ve daha onceden tanimlanmis gen dizileri ile karsilastirilarak direnc belirlenmistir. Bulgular: Calismamizda degerlendirilen toplam 20 influenza A (H1N1) izolatinin iki (%10)’sinde oseltamivir direncinin gostergesi olan 274. (N1 numaralandirmasinda 275) aminoasitte histidinden (H) tirozine (Y) degisim saptanmistir. NA segmentinin kismi dizi analizi sonuclari National Center for Biotechnology Information (NCBI) GenBankasinda GQ369800, GQ369799, GQ369798 giris numaralari ile sunulmustur. Sonuc: NA dizi analizi ile ulkemiz orta ve dogu kisimlarindan gelen influenza A (H1N1) suslarinin H274Y oseltamivir direncinin degerlendirilmesiyle Turkiye’de 2008 yilinda noraminidaz inhibitorlerine direncin gorulmeye baslandigini belirlenmistir. Anahtar Sozcukler: Influenza A (H1N1), oseltamivir direnci, H274Y mutasyonu, Turkiye The NA genes of influenza, viruses were sequenced. The presence of resistance was inferred by comparison with published sequences and known resistant mutations. Results:In our study, the histidine (H) to tyrosine (Y) substitution at position 274 (275 in N1 numbering) of the NA gene was evaluated. The change indicating resistance to oseltamivir was observed in two isolates of the total 20 influenza A (H1N1) strains (10%). The partial sequence analysis results of NA segments were submitted to the National Center for Biotechnology Information (NCBI) GenBank with accession numbers GQ369800, GQ369799, GQ369798. Conclusion: Resistance to neuraminidase inhibitors was determined by NA gene sequencing in the isolates of A/H1N1 strains taken from central and eastern parts of Turkey by monitoring the presence of H274Y oseltamivir-resistant in 2008.

Detection of Oseltamivir-Resistant Influenza A (H1N1) Viruses with H274Y Mutation during the 2007 - 2008 Influenza Season in Turkey