Genetic analysis of post-pandemic influenza A(H1N1)pdm09 haemagglutinin virus variants that caused mild, severe and fatal infections in N. A. Melidou*, M. Exindari, G. Gioula, N. Malisiovas 2nd Laboratory of Microbiology, Medical School, Aristotle University of , Greece National Influenza Centre for Northern Greece

NS75P,N129D,N287S A//416/2011 A/Edessa/284/2011 95 A/Thessaloniki/532/11 A/Thessaloniki/644/11 Introduction A/Thessaloniki/1151/11 A//1246/11 A//1524/11 • Genetic analysis of post-pandemic influenza A(H1N1)pdm09 strains isolated in northern Greece A/Thessaloniki/555/11 A/Thessaloniki/446/11 A/Thessaloniki/118/11 • Molecular analysis of HA1 sequences to identify variations and any possible relation to severity of illness. A/Kavala/469/11 A/Thessaloniki/1241/11 A//803/11 • Comparison with data obtained during the pandemic period. A/Thessaloniki/1378/11 A/Giannitsa/1052/11 A/Thessaloniki/1168/11 A/Thessaloniki/1376/11 A//271/11 N56S A/Kavala/597/11 65 A/Thessaloniki/539/11 A/Halkidiki/606/11 A/Alexandroupolis/1218/2011 Materials and methods A/Thessaloniki/63/11 A/Thessaloniki/515/11 S84I A/Serres/308/11 63 A/Thessaloniki/43/11 • 1870 pharyngeal swabs - RNA extraction (Qiagen viral RNA mini kit) A//807/11 I5V A/Thessaloniki/599/11 64 A/Veroia/525/11 • Real time RT-PCR for detection of A(H1N1)pdm09 influenza (CDC protocol) - HA1 gene of 102 strains sequenced K171R A/Kavala/470/11 65 A/Giannitsa/414/11 A/Thessaloniki/537/2011 • Phylogenetic analysis: MEGA 5.1, ClustalW, GISAID. A/Thessaloniki/1040/11 A/Thessaloniki/503/11 A/Kavala/19/11 A/Thessaloniki/604/11 A/Thessaloniki/678/11 A/Thessaloniki/983/11 A/Thessaloniki/616/11 7 A/Thessaloniki/570/11 A/Thessaloniki/451/11 Results A/Alexandroupolis/815/11 Group 6 A/Thessaloniki/1120/11 A/Kavala/564/11 •The D222G variation was observed only in fatal cases (6). None of the examined strains had the Q293H variation. A/Thessaloniki/329/11 A/Thessaloniki/74/11 A/Thessaloniki/654/11 •N-linked glycosylation sites: two 2010-11 strains had thesubstitution N287S, but this did not change the neutral and A/Thessaloniki/774/11 A/Thessaloniki/151/11 A/Thessaloniki/407/11 polar properties of the side chain. Interestingly, the same sites was only altered in two 2009-10 circulating strains. A//1123/11 A//785/2011-M • A/Thessaloniki/712/11 Most viruses clustered with group 6 viruses, whereas a number of viruses clustered with groups 3,4,5 and 7. None A/Thessaloniki/287/11 A/Thessaloniki/516/11 clustered with groups 1,2 or 8. A/Kilkis/533/11 A/Veroia/321/11 A/Thessaloniki/410/11 •Genetic analysis showed that the HA amino acid sequences were largely similar to the vaccine strain, with sequence A//199/2011 D97N, 21 A/Alexandroupolis/1290/11 S185T A/Thessaloniki/164/11 similarity of 96-99%. A/Thessaloniki/677/11 A/Thessaloniki/506/11 • A/St Petersburg/27/11 Certain signature amino acid changes, such as P83S, D97N, S185T, S203T and I321V, persisted from the pandemic V152I A/Thessaloniki/839/11 63 A/Thessaloniki/1391/11 period, indicating that they offer some selective advantages to the virus. A/Alexandroupolis/1219/11 65 A/Thessaloniki/1541/2011 A/Thessaloniki/508/11 •Interestingly, some persistent variations were observed at amino acid residues in the vicinity of antigenic or receptor A/Thessaloniki/605/11 A/Thessaloniki/73/11 A/Katerini/199/11 binding sites, such as I116V, S143G, V152I, K171R, S185T, S203T, D223R. 18 A/Thessaloniki/672/11 Influenza Season 2010-2011 A//1705/2011 A/Thessaloniki/447/11 Total examined 1870 A/Thessaloniki/1329/11 A/Thessaloniki/174/11 A/Thessaloniki/543/11 Total positive samples 848(45.3%) 65 A/Serres/12/11 G170R A/Thessaloniki/1167/11 Positive influenza A(H1N1) 821 (96.8%) 54 A/Alexandroupoli/1349/11 A/Giannitsa/596/11 Positive influenza A(H3N2) 13 (1.53%) A/Thessaloniki/657/11 A/Komotini/261/11 18 A/Thessaloniki/385/11 Positive influenza B 14 (1.65%) 10 D97N,R205K, A/Thessaloniki/163/11 Conclusions I216V,V249L A/Kavala/464/11 Fatal cases 49 88 A/Thessaloniki/268/11 A/Astrakhan/1/11 87A/Thessaloniki/1164/11 5  High risk groups were considered to be pregnant women and obese individuals, whereas the elderly were much less A/Alexandroupoli/204/11 A/Alexandroupolis/819/11 A197T, A/Thessaloniki/607/11 affected than expected, probably due to the extensive vaccination of this age group and to the increased level of pre- 15 S143G, A/Xanthi/265/11 S185T A/Thessaloniki/487/11 44 A/St Petersburg/100/11 existing antibodies that were cross-reactive with the pandemic influenza virus. However, the median age of severe and 53 A//552/11 A/Kavala/463/11 47 A/Alexandroupoli/827/11 71 fatal cases was higher, 44 and 54 years during the post-pandemic period, compared to 47 years during the pandemic. 16 A/Serres/663/11 7 A//456/11 A/Naousa/626/11  Overall the 2010-2011 circulating strains had a close match with the vaccine strain A/California/7/2009, with nucleotide 19A/Serres/1244/11 A186T,N14 A/Alexandroupoli/530/11 6D,T147K, 3 A/Florina/743/11 similarity between 96-99%, whereas in 2009-10 between 98-99.7%. V193A A/Stockholm/13/11 A/Ghana/601/11 69 8 60 A/Norway/2552/12  There is an obvious trend of the new virus strains to accumulate amino acid changes on their antigenic sites and form A/Czechrepublic/32/11 A/Thessaloniki/2812/09 5 A/Kilkis/557/11 A//111/11 new phylogenetic clades. The coexistence of different clusters indicated co-circulation of lineages in northern Greece. N125D58 5 A/Thessaloniki/1099/11 4 57 A/Thessaloniki/1078/11  Epidemiological analysis revealed that the post-pandemic period 2010-11 was a severe influenza season with increased A/Christchurch/16/10 16 A/Kastoria/245/10 7 A/Thessaloniki/2502/09 morbidity and mortality rates, even though exact rates cannot be determined because of the increased number of A/Thessaloniki/4183/09 A/Thessaloniki/225/10 6 A/Lviv/N9/09 severe cases that are tested at the NIC. A/Alexandroupoli/1480/11 A/Hong kong/2212/10 18 I295V, 68 A//Hong kong/3934/11 3  Molecular analysis revealed a number of variations at the HA1 sequences of northern Greek circulating strains. A134T, 64 A/Kavala/324/11 S183P 66 A/Thessaloniki/453/11 S203T A/Thessaloniki/213/10  36 A/Kastoria/2636/09 Constant epidemiological and molecular surveillance is important to monitor the efficacy of the A/Kavala/219/10 90 A/Thessaloniki/228/10 vaccine and assess the severity of each influenza season. A/Auckland/3/09 P83S, 22 A/England/195/09 I321V A/Bayern/69/09 A/California/7/09 - VACCINE STRAIN

0.002 Bold Severe/Fatal