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Volume 4, Issue 6(Suppl) J Infect Dis Ther
ISSN: 2332-0877 JIDT, an open access journal
Page 48
Influenza 2016
September 12-13, 2016
conferenceseries.com
Influenza
September 12-13, 2016 Berlin, Germany
2nd International Conference on
H1N1 2009 pandemic influenza virus: Kinetic, structural and thermodynamic analysis of the
H275Y, I223V and S247N neuraminidase resistant mutants
Jana Pokorna1, Kozisek Milan1, Pachl Petr1, Rezacova Pavlina1, Machara Ales2, Carlos Berenguer Albinana2, Karlukova Elena1, 2, Hejdanek Jakub1, 2, Pisackova
Anezka1, 2 and Konvalinka Jan1, 2
1Academy of Sciences of the Czech Republic, Czech Republic
2Charles University, Czech Republic
Influenza is an acute viral infection that can cause serious complications and death, especially among elderly individuals and
patients at risk. Neuraminidase, which plays an essential role in virus replication, is the main influenza drug target. At present,
two neuraminidase inhibitors (NAIs) are licensed worldwide for therapeutic and prophylactic uses (oseltamivir marketed as Tamiflu
and zanamivir, Relenza) and two others have been authorized in various countries for the emergency treatment during pandemics.
However, drug resistant viruses readily emerge because of the high mutation rate of their RNA dependent RNA polymerase. Indeed,
resistance to oseltamivir, the most prescribed NAI was detected not only during treatment and prophylaxis but also in influenza
virus variants in untreated individuals. Novel neuraminidase inhibitor resistance substitutions I223V and S247N alone or in
combination with a major oseltamivir resistance mutation H275Y have been observed recently in the 2009 pandemic H1N1 viruses.
We overexpressed the ectodomain of the wild type neuraminidase from the influenza virus A/California/07/2009 (H1N1) as well
as recombinants containing H275Y, I223V and S247N single mutation and the H275Y, I223V and H275Y, S247N double mutants
in Drosophila Schneider S2 cells and purified them by one-step purification using a streptavidin derivative. In order to quantify the
level of resistance we enzymologically characterized these enzymes with the set of in-house designed and synthesized derivatives of
oseltamivir. Thermodynamic analyses of oseltamivir binding to neuraminidase monomutants were performed by protein micro-
calorimetry. Finally, we crystallized neuraminidase variants in complexes with oseltamivir to structurally explain the resistance
mechanism.
Biography
Jana Pokorna has completed her PhD in Biochemistry from Charles University in Prague in 2013. She is working as a Postdoctoral Fellow at the Institute of Organic
Chemistry and Biochemistry ASCR, v.v.i. Her research interests are activity, inhibition, drug and resistance development focusing on HIV protease and neuraminidase from
the influenza virus. She has published 11 peer viewed papers and she is the author of 3 patents.
Jana Pokorna et al., J Infect Dis Ther 2016, 4:6(Suppl)