Detection of Neuraminidase Inhibitor Resistant Influenza A/H3N2 Viruses by Nucleotide Sequencing Method

Document Type : Original Article


1 Department of Virology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.

2 Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

3 Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.


Background and Objective: Influenza A viruses are the major cause of epidemics and pandemics worldwide. Many researches are underway in order to prevent and treat. Recently Oseltamivir (Tamiflu), a neuraminidase inhibitor (NAI), has been used for their treatment and prevention. The pitfall of using antiviral drugs is emergence of resistant mutants. Different researches in enzyme active site mutation analysis of NAI resistant mutants revealed that common mutation sites are located at 119, 274, 292 residues. The aim of this study was detection of R292K and H274Y mutations in circulating influenza A/H3N2 viruses by using nucleotide sequencing method.
Subjects and Methods: In order to amplify NA gene mutation sites, we used conventional RT-PCR assay on 50 influenza A/H3N2 isolates and then PCR products were sequenced. Evaluation and analysis of raw data by Bioedit version (2011) software was performed.
Results: Evaluation of nucleotide sequences did not show any isolates with R292K and or H274Y mutations and all the examined viruses were sensitive to NAI.
Conclusion: The findings of this study demonstrate that circulating A/H3N2 viruses are sensitive to NAI and lack resistant mutation sites. As detection of drug resistant mutants with high sensitivity and specificity is very important for efficient treatment strategies, we suggest nucleotide sequencing as a choice method in laboratories protocol because of its accuracy, capability and high efficiency for monitoring of influenza drug resistant mutants in population.


Yavarian J, Mokhtari Azad T, Nadji SA, Zeraati H, M.Naseri. Analysis of the   hemagglutinin and neuramindiase genes of human influenza A/H3N2 viruses  circulating in Iran between 2005 and 2007: antigenic and phylogenetic relationships to vaccine strains. Intervirol 2010; 53: 133-140.
2-Yavarian J, Mokhtari Azad T,Shafiei Jandaghi NZ, Nategh R. Amantadine-resistant influenza A (H3N2) viruses in Iran. Acta Virologica  2009; 53: 135-138.
3-Chutinimitkul S, Suwannakarn K, Chieochansin T, Mai le Q, Damrongwatanapokin S, Chaisingh A, et al. H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes. J VIROL METHODS 2007 Jan;139(1): 44-9.
4-Carr MJ, Sayre N, Duffy M, Connell J, Hall WW. Rapid molecular detection of the H275Y oseltamivir resistance gene mutation in circulating influenza A (H1N1) viruses. J VIROL METHODS 2008 Nov; 153(2): 257-62.
5-Redlberger-Fritz M, Aberle SW, Strassl R, Popow-Kraupp T. Rapid identification of neuraminidase inhibitor resistance mutations in seasonal influenza virus A(H1N1), A(H1N1)2009, and A(H3N2) subtypes by melting point analysis. Eur J Clin Microbiol Infect Dis 2012 Jul; 31(7): 1593-601.
6-McKimm-Breschkin JL. Influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. Influenza Other Respir Viruses 2013 Jan;7 Suppl 1: 25-36.
7-Chidlow GR, Harnett GB, Williams SH, Tempone SS, Speers DJ, Hut AC, “et al’. The detection of oseltamivir-resistant pandemic influenza A/H1N1 2009 viruses using a real-time RT-PCR assay. J VIROL METHODS 2010 Oct; 169(1): 47-51.
8-Anton A, Lopez-Iglesias AA, Tortola T, Ruiz-Camps I, ‘et al’. Selection and viral load kinetics of an oseltamivir-resistant pandemic influenza A (H1N1) virus in an immunocompromised patient during treatment with neuraminidase inhibitors. DIAGN MICR INFEC DIS 2010 Nov; 68(3): 214-9.
 9-Smith JR, Rayner CR, Donner B, Wollenhaupt M, Klumpp k, Dutkowski R. Oseltamivir in seasonal, pandemic, and avian influenza: a comprehensive review of 10-years clinical experience. ADV THER. 2011 Nov; 28(11): 927-59.
10-Zambon MC. Surveillance for antiviral resistance. Influenza Other Respir Viruses 2013 Jan; 7 Supp l 1: 37-43.
11- Okomo-Adhiambo M, Sheu TG, Gubareva LV. Assays for monitoring susceptibility of influenza viruses to neuraminidase inhibitors. Influenza Other Respir Viruses. 2013 Jan; 7 Suppl 1: 44–49.
12- Van der Vries E, Anber J,  van der Linden A, Wu Y, Maaskant J, Stadhouders R, ‘et al’. Molecular Assays for Quantitative and Qualitative Detection of Influenza Virus and Oseltamivir Resistance Mutations. J Mol Diagn  2013 May; 15(3): 347–354.
13-Cheng TJ, Wang SY, Wen WH, Su CY, Lin M, Wu HS, ‘et al’. Chemical probes for drug-resistance assessment by binding competition (RABC): oseltamivir susceptibility evaluation. Angewandte Chemie 2013 Jan; 52(1): 366-70.
14- Hurt A C, Chotpitayasunondh T, Cox N I,  Daniels R, Fry A M, Gubareva V L, ‘et al’.  Antiviral resistance during the 2009 influenza A H1N1 pandemic: public health, laboratory, and clinical perspectives. Lancet Infect Dis 2012 Mar; 12(3): 240-248.
15- Rameix-Welti M A, Munier S, Lee Gal S, Cuvelier F, Agou F, Enouf V, ‘et al’. Neuraminidase of 2007-2008 influenza A(H1N1) viruses shows increased affinity for sialic acids due to the D344N substitution. Antivir Ther 2011; 16(4): 597-603.