Challenges in the antigenic characterization of circulating influenza A(H3N2) viruses. during the influenza season: an ongoing problem?

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1 JCM Accepted Manuscript Posted Online 18 February 2015 J. Clin. Microbiol. doi: /jcm Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 2 Challenges in the antigenic characterization of circulating influenza A(H3N2) viruses during the influenza season: an ongoing problem? Athanasios Kossyvakis 1*, Vasiliki Pogka 1*, Aggeliki Melidou 2, Afroditi Moutousi 1, Georgia Gioula 2, Antonios Kalliaropoulos 1, Maria Exindari 2, Mary Emmanouil 1, Elina Horefti 1, Georgia Spala 3, Adam Meijer 4, Nikolaos Malisiovas 2, Andreas F. Mentis 1#, 1 National Influenza Reference Laboratory for Southern Greece, Hellenic Pasteur Institute, Athens, Greece, 2 National Influenza Reference Laboratory for Northern Greece, Medical School, Aristotle University of Thessaloniki, Greece, 3 Department of Epidemiological Surveillance and Intervention, Hellenic Centre for Disease Control and Prevention, Athens, Greece, 4 National Institute for Public Health and the Environment, Centre for Infectious Disease Control, Bilthoven, Netherlands Running Head: Flu A(H3N2) antigenic characterization challenges #Address correspondence to Dr Andreas F. Mentis, mentis@pasteur.gr * These authors contributed equally to this manuscript Abstract word count: 143 Text word count:

2 25 Abstract Genetic and antigenic characterization of 37 representative influenza A(H3N2) strains isolated in Greece during the winter season was performed to evaluate matching of the viruses with the seasonal influenza A/Perth/16/2009 vaccine strain. Haemagglutinin-gene sequence analysis revealed that all Greek strains clustered within the Victoria/208 genetic clade. Furthermore, mutations in the antigenic and glycosylation sites suggested potential antigenic drift. Our haemagglutinationinhibition (HI) analysis showed that the Greek viruses were Perth/16-like, however, these viruses were characterised as Victoria/208-like when tested at the UK WHO Collaborating Centre (WHOCC) by HI in the presence of oseltamivir, a finding consistent with the genetic characterization data. Variability in the HI testing performance experienced by other European laboratories indicated that A(H3N2) virus antigenic analysis has limitations and until its standardization, national influenza reference laboratories should include genetic characterization results for selecting representative viruses for detailed antigenic analysis by WHOCC. Introduction Influenza virus is an important causative agent of respiratory tract infections and annual influenza epidemics have a significant impact on global public health (1). There are two main types of influenza virus infecting humans, namely A and B. Influenza A viruses can be subtyped according to the antigenic properties of their haemagglutinin (HA) and neuraminidase (NA) surface glycoproteins (2, 3). Currently circulating subtypes of seasonal influenza A viruses are A(H1N1)pdm09 and A(H3N2). 2

3 Vaccination is the major protective barrier against outbreaks of influenza respiratory disease. However, seasonal circulating influenza viruses exploit genetic instability of the viral HA to escape the immune response and avoid matching with homologous vaccine viruses, thus forcing annual evaluation of vaccine effectiveness (4). The HA glycoprotein mediates viral entry into host cells and is the major antigenic target for neutralizing antibodies induced by vaccines. Host cell proteases cleave HA into two domains, HA1 (the unique component of the viral HA globular head) and HA2 (the stalk and anchor in the cell membrane) (5). The HA1 domain contains the receptor-binding cavity as well as major antigenic sites of the HA molecule. Acquisition of mutations within this region has therefore the greatest effect on viral antigenic structure. Seasonal influenza virus A(H3N2) is a typical example of vaccine escape virus. Since the H3N pandemic, 108 amino acid changes clustered at 63 residue positions in HA1 resulting in twenty seven A(H3N2) strain alterations in the vaccine formulation, twice as often as for the other vaccine components, influenza B or former seasonal A(H1N1) virus (6). The majority of these changes clustered into the viral antigenic sites, which have been previously named as A to E antigenic sites in A(H3N2) viruses (7-9). Previous genetic analysis of Greek influenza A(H3N2) viruses from 2004 through 2008 has indicated the emergence of variations at antigenic and glycosylation sites of the HA1 domain (10). Genetic analysis of the viral HA1 domain and antigenic analysis of viruses are important tools for identification of circulating viruses with characteristics that might lead to vaccine failure. Both laboratory-based activities are carried out by the National Influenza Reference Centers (NICs) and the WHO Collaborating Centers for Reference and Research on Influenza (WHO CCs) in order to examine the molecular 3

4 evolution of influenza virus and its impact on antigenic characteristics, thus contributing to the evaluation of each year s vaccine and recommendations for the following year s vaccine candidates (11). The aim of the present study was the molecular characterization and phylogenetic analysis of representative influenza A(H3N2) strains that circulated in Greece during peak influenza activity of , as well as the identification of antigenic and genetic variations when compared with the vaccine and other reference strains. Materials and methods During the winter season, the presence of influenza virus RNA was detected by multiplex real-time RT-PCR in 890 out of 1806 (49.3%) respiratory specimens from patients with influenza-like illness (ILI), 441 (24.4%) out of which were positive for influenza A(H3N2) virus while the remaining 449 (24.9%) specimens were found positive for influenza B virus. The first and the last positive A(H3N2) case was confirmed on January 8 th (week 1) and April 18 th (week 16) respectively. During the same period only a single case positive for A(H1N1)pdm09 was confirmed. The influenza virus A(H3N2) strains included in the study were selected from the A(H3N2) positive group according to criteria recommended by WHO for selection of sufficient and representative viruses for characterization studies and shipment to WHO CC, Mill Hill (London, UK) (12). In total, 37 influenza A(H3N2) positive specimens with high viral load (PCR Ct value <30) were selected, accounting for approximately 10% of the A(H3N2) positive specimens. Eighteen of them were selected amongst specimens sent by the sentinel physicians to ensure spatio-temporal 4

5 representativeness. The remaining 19 originated from hospitalized patients or intensive care unit (ICU) cases. Five to seven specimens were selected from each of the 0-1, 2-5, 6-18, 19-35, and >65 age groups. Sixteen (43%) of the total 37 specimens were selected until the end of January, while the remaining 21 (57%) specimens were collected during progression of the influenza outbreak till April (Figure 1). Sequence and phylogenetic analysis For sequence analysis, a 1096 bp DNA fragment of the HA1 domain was amplified directly from the clinical samples by an in-house nested PCR protocol. Primer sequences for PCR and sequencing had been published by WHO CC in the European Influenza Surveillance Network (EISN) protocol library with restricted access among the NICs. The resulting amplicons were purified utilizing the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and the MinEluteTM gel extraction kit (Qiagen, Hilden, Germany) and sequenced in both directions using the GenomeLab DTCS-quick start sequencing kit (Beckman Coulter, Brea, California) on a CEQTM 8000 genetic analyzer (Beckman Coulter, USA). All sequences obtained in this study were deposited into GISAID (Global Initiative on Sharing All Influenza Data) under accession numbers EPI354678, EPI EPI354689, EPI358885, EPI358887, EPI358889, EPI358891, EPI358893, EPI358945, EPI368826, EPI368828, EPI369758, EPI369786, EPI EPI359560, EPI475780, EPI475800, EPI475811, EPI475813, EPI EPI Multiple sequence alignment of the obtained sequences and influenza reference strains retrieved from GenBank and GISAID was performed using the BioEdit sequence alignment editor ( Phylogenetic analysis was done using the molecular evolutionary genetics analysis 5

6 (MEGA) program, version 5 (13). The neighbor-joining method (NJ, tree algorithm inferred with the Kimura 2-parameter substitution model of sequence evolution) (14, 15) was used to construct phylogenetic trees, and a bootstrap resampling analysis was performed (1.000 replicates) to test tree reliability (16). In order to identify the presence of new N-linked glycosylation sites, the above sequences were also screened for the consensus asparagine-x serine/threonine (NXS/T) motif (X is any amino acid except proline). Antigenic analysis All 37 A(H3N2) strains included in the study were inoculated in MDCK SIAT-1 cells. Thirty of them had sufficient titer and were further compared to the A/Perth/16/2009 seasonal influenza H3N2 vaccine component for antigenic relatedness by haemagglutination-inhibition (HI) method according to WHO recommendations (17). For the HI testing, a reference ferret antiserum raised against the egg grown vaccine virus A/Perth/16/2009, kindly provided by the WHO CC in UK, was used along with guinea pig red blood cells (RBC) (1% v/v standardized solution) obtained from the animal unit of the Hellenic Pasteur Institute. The latter reference antiserum was treated with receptor-destroying enzyme according to WHO CC standard procedure. To denote a virus isolate as being like a vaccine/reference virus it s HI titer with the reference ferret antiserum should differ by no more than 4-fold (usually a decrease), in a 2-fold dilution series, compared to the HI titer with the vaccine/reference virus itself. The HI titers were the reciprocals of the highest dilution at which virus binding to guinea pig RBC was blocked. Further detailed antigenic characterization was performed on a subset of the above strains (N=12) by the WHO CC in UK. These strains were selected at the 6

7 beginning phase of the seasonal epidemic and were of extensive genetic variation as demonstrated by the HA sequence analysis (Figure 2). HI analysis by WHO CC was performed using the above reference antiserum and other ferret antisera raised against cell-grown representative European strains. The HI protocol utilized by the WHO CC included the addition of 20 nm oseltamivir carboxylate in the assay plate to prevent haemagglutination by binding through the neuraminidase. Preliminary testing in our laboratory to implement HI analysis in the presence of oseltamivir carboxylate according to WHO CC standard procedure resulted in a very low haemagglutination titer that prevented using them for HI assay. Results Sequence and Phylogenetic analysis Phylogenetic analysis indicated clustering of the Greek strains within genetic groups 3A (46.0%), 3B (35.1%), 3C (10.8%) and 6 (8.1%), all belonging to the Victoria/208 clade (Figure 2). Genetic groups were assigned by the WHO CC in UK genetic studies (18). All A(H3N2) sequences revealed an HA nucleotide identity of % and a deduced amino acid identity of % with each other. The above strains were also compared with the influenza season vaccine strain for the Northern hemisphere, A/Perth/16/2009, and presented with a nucleotide identity of % and a deduced amino acid identity of %. Interestingly, none of the Greek strains clustered with vaccine strain A/Perth/16/2009 in the same genetic clade (Perth/16 clade). HA1 sequencing of the A(H3N2) strains also revealed in total 19 amino acids variations at the five viral antigenic sites A, B, C, D and E compared with the vaccine strain A/Perth/16/2009 (Table 1). Strains belonging to group 3A 7

8 accumulated 2 amino acid variations at antigenic site A, 3 at D and 1-2 at E; strains belonging to group 3B possessed 2 amino acid variations at antigenic site A, 1 at B, 0-2 at C, 3 at D and 1 at E; strains belonging to group 3C accumulated 1 amino acid variation at antigenic site A, 1 at B, 4 at C, 3 at D and 1 at E; finally, strains belonging to group 6 revealed 1 amino acid variation at antigenic site A, 2 at C, 3-4 at D and 2 at E. The most divergent strain, A/Athens/16/2012, belonged to group 3C and revealed the emergence of 4 amino acid substitutions in 2 different antigenic sites, C and D respectively. Following the molecular analysis of HA1 of circulating strains, variations were also observed in the N-linked glycosylation sites. Substitution of lysine (K) with aspartic acid (D) at position 144 in 17 Greek strains, all belonging to the 3A group, resulted in the loss of a glycosylation site. In contrast, substitution of lysine (K) with asparagine (N) at the same position in 20 other strains, belonging to groups 3B, 3C and 6, resulted in the gain of a glycosylation site. In the 4 strains belonging to group 3C, substitution of serine (S) with asparagine (N) at position 45 resulted in a gain of another glycosylation site (Table 1). No particular segregation of sequences according to epidemic timeline (beginning-peak-end of ILI consultation rates) was observed (not shown). All strains belonging to groups 3A, 3B and 6 circulated during the peak of the season (weeks 4 to 11, 3 to 10 and 4 to 10, respectively), whereas strains belonging to group 3C were detected 2-3 weeks earlier (week 1 to 10) but co-circulated with the other groups from week 3/2012 onwards. Viral strains identified from ICU cases, as well as the A/Athens.GR/369/2012 viral strain originating from the single fatal case included in the study, were closely related to the other Greek strains examined at the genetic level (Figure 2). Finally, all 8

9 strains allocated to the 3C genetic group were obtained from hospitalized patients and exhibited more amino acid alterations at antigenic and glycosylation sites in comparison with the rest of the viruses. Antigenic analysis HI testing without oseltamivir revealed that 19 out 30 viruses (63.3%) had an HI titer of 1:640 and the remaining 11 an HI titer of 1:320 against the reference antiserum raised against egg propagated strain A/Perth/16/2009, whilst the homologous reaction of the vaccine egg propagated strain A/Perth/16/2009 with the same reference antiserum had a titer of 1:1280. Thus, all 30 virus isolates showed not more than 4-fold reduction in HI reactivity compared to the vaccine reference virus and therefore they were characterized as A/Perth/16/2009-like, despite phylogenetic allocation of these viruses to the Victoria/208 clade. Twelve viruses representing genetic variation within groups 3A, 3B and 3C of the Victoria/208 clade, were further analyzed by the WHO CC in the UK (Table 2). In contrast to our results, antigenic analysis in HI testing with oseltamivir illustrated poor reactivity with the reference antiserum raised against egg propagated strain A/Perth/16/2009, with titers 8-fold reduced compared with the homologous titer, suggesting that the 12 viruses were indeed antigenically not A/Perth/16/2009-like. However, in accordance with our phylogenetic analysis, all isolates exhibited <4-fold titer reduction when compared with cell-propagated reference viruses of the Victoria/208 clade which were isolated in Europe. 217 Discussion Antigenic characterization supported by gene sequencing of HA and NA genes of influenza viruses performed by NICs is the cornerstone of influenza risk 9

10 assessment and the development of seasonal influenza vaccines. Selection of representative strains for testing is a critical step for this task. We selected 37 A(H3N2) viruses from the winter influenza season for characterization amongst a large number of clinical specimens positive for A(H3N2) virus. The selection was based on the criteria recommended by WHO to select to the possible extent sufficient and representative viruses for characterization studies and shipment to WHO CC (12). Sixteen (43%) of the above specimens were selected until the end of January, since the timely genetic and antigenic characterization of the viruses isolated at the beginning of the influenza season as well as their prompt shipment to WHO CC for further characterization are critical for the WHO consultation on the Northern Hemisphere vaccine composition in February. However, the percentage of specimens required for virus isolation and characterization is not addressed in the WHO guidelines. Each NIC laboratory should choose an algorithm based on the WHO selection criteria that best fits laboratory test flow, surveillance needs, resources and laboratory capacity. Phylogenetic analysis of the influenza A(H3N2) viruses examined during the winter season in Greece revealed the circulation of viral strains that did not cluster in the Perth/16 clade, represented by the A/Perth/16/2009 vaccine strain, but to 4 different genetic groups within the Victoria/208 clade and predominantly to group 3 (91.9%). During the same period, all countries within the European Influenza Surveillance Network also reported the circulation of A(H3N2) viruses genetically different from the vaccine strain (19, 20). The majority of those strains belonged to group 3 of the Victoria/208 clade. Each one of the examined strains displayed at least 4 alterations in 3 antigenic sites, with the most divergent strain, A/Athens/16/2012, possessing 4 amino acid 10

11 substitutions in 2 different antigenic sites. Genetic analysis of the HA1 domain is important as it has been shown that amino acid changes within the antigenic sites can significantly affect the antigenic properties of influenza viruses and cumulatively enhance antigenic drift (5, 21, 22). Particularly interesting are amino acid substitutions in antigenic sites localized around the receptor binding pocket, like sites A and B. It is noteworthy that the haemagglutinin of antigenically distinct viruses of epidemic significance has a mutation in the region of the antigenic site A (7, 8, 21). The K144N/D substitution has been confirmed in all A(H3N2) Greek strains examined. The majority of the strains also contained the substitution N145S within this region. Moreover, HA1 amino acid sequence analysis showed an altered potential N-linked glycosylation site at amino acid 144 in all Greek strains. Amino acid alterations within the relative conserved N-linked glycosylation sites of the viral HA may alter the antigenicity and virulence of influenza virus by shielding the major antigenic epitopes (23). The above genetic variations indicated potential antigenic drift for all the strains examined. Despite that, preliminary antigenic characterization performed by our laboratory did not show that emerging genetic variants were antigenically different from the A/Perth/16/2009 vaccine strain. Similarly, all European countries reported the circulation of Perth/16-like viruses by antigenic characterization (24). In detailed antigenic studies carried out by the WHO CC, Greek strains demonstrated reduced reactivity ( 8-fold) with ferret antiserum raised against the egg grown vaccine virus A/Perth/16/2009. These studies, performed by HI testing in the presence of oseltamivir carboxylate, efficiently detected the antigenic variants, and were in agreement with the genetic characterization studies (25). It seems that, apart from HA, viral NA of Α(Η3Ν2) viruses also interacts with the erythrocytes used in the HI 11

12 testing and to circumvent this interaction, Lin et al. recommended that HI assays should be carried out in the presence of oseltamivir (26). The agglutination of RBCs by the viral NA has been shown to be the result of a mutation in aspartic acid 151 of NA to glycine, asparagine, or alanine (26). NA gene sequence analysis of the 12 Greek isolates was carried out by WHO CC (data not shown). Interestingly, no neuraminidase receptor binding variants of Greek influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site were confirmed. In contrast to WHO CC findings, the addition of oseltamivir in our HI testing prevented the antigenic assessment of HA viral protein due to low levels of hemagglutination titers. The overall reduced sensitivity of our MDCK SIAT-1 cells compared to WHO CC MDCK SIAT-1 cell culture, the level of optimization of our HI testing in the presence of oseltamivir and slight differences in cell lines, guinea pig red bloods cells and batches of oseltamivir neuraminidase inhibitor could account for the resultant assay limitations. It is noteworthy that significant variability in the HI testing using oseltamivir has been reported recently by WHO (27). Moreover, the use of the provided ferret antiserum raised against the egg grown vaccine virus to antigenically characterize cell-grown H3N2 viruses may be of additional concern. It is known that when a human influenza virus is adapted to growth in eggs, it undergoes phenotypic changes that might include changes to its antigenicity/immunogenicity (28). Nevertheless, the evolving nature of H3N2 HA receptor binding necessitated the addition of oseltamivir even when ferret antisera raised against cell-grown viruses were used in the HI testing (26). Unlike the influenza season included in the study, during the following and influenza seasons both genetic and antigenic testing without oseltamivir were effective to monitor Greek strains fit to the respective 12

13 seasonal vaccine strains. Other European countries also reported that circulating viruses were genetically and antigenically similar to the vaccine strains (29, 30). The above findings may be explained by the fact that the H3N2 viruses were antigenically conserved. If circulating viruses of the and influenza seasons had exhibited antigenically significant genetic variation, the HI assay without oseltamivir could misinterpret agglutination patterns and miss out antigenic drift. Difficulties in the HI testing re-emerged during the influenza season due to failure of a proportion of the circulating H3N2 viruses to agglutinate guinea pig or other red blood cells (John McCauley-WHO CC director, personal communication, 19 December 2014). Nonetheless, challenges experienced in the antigenic characterization of the H3N2 viruses by HI testing with or without oseltamivir necessitates HI assay standardization. Until that, national influenza reference laboratories should not rely solely on HI-assay results for selecting representative viruses for detailed antigenic analysis by WHO CC, but should include genetic characterization results in that assessment. Timely detection of mutations within the antigenic sites facilitates the selection of interesting and risky viruses that should be sent to the WHO CC for detailed antigenic characterization. In this way, the shipment of numerous specimens and WHO CC overload prior to the vaccine recommendation decisions could be avoided. Boosting of genetic analysis would acquire timely sharing of related metadata, both clinical and epidemiological. This emerging requirement for vaccine virus selection could be fulfilled through submission of genetic data to genetic databases such as GISAID's publicly accessible platform. Increased difficulties in antigenic characterization, might be overcome by the integration of technical improvements such as the use of erythrocyte substitutes to 13

14 select exclusively for the viral HA glycoprotein (27). Even most promising is the identification of antigenic variants using sequence data alone based on highthroughput genetic sequencing combined with advanced bioinformatics tools (31). However, at the present time, traditional methods should not be discontinued. Antigenic characterization limitations have not been observed for influenza B viruses and other influenza type A viruses like A(H1N1)pdm09 virus and NICs should continue virus propagation and HI testing for these viruses. Moreover, virus isolates, including H3N2, are needed not only for antigenic characterization studies but also for other NIC activities like phenotypic antiviral susceptibility testing and probably highthroughput sequencing techniques. Downloaded from on November 25, 2018 by guest 14

15 References 1. Fiore AE, Uyeki TM, Broder K, Finelli L, Euler GL, Singleton JA, Iskander JK, Wortley PM, Shay DK, Bresee JS, Cox NJ Prevention and control of influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP), MMWR Recomm Rep 59: Gamblin SJ, Skehel JJ Influenza hemagglutinin and neuraminidase membrane glycoproteins. J Biol Chem 285: Medina RA, Garcia-Sastre A Influenza A viruses: new research developments. Nat Rev Microbiol 9: WHO. Weekly epidemiological record. Vaccines against influenza. WHO position paper Nov Shih AC, Hsiao TC, Ho MS, Li WH Simultaneous amino acid substitutions at antigenic sites drive influenza A hemagglutinin evolution. Proc Natl Acad Sci U S A 104: Popova L, Smith K, West AH, Wilson PC, James JA, Thompson LF, Air GM Immunodominance of antigenic site B over site A of hemagglutinin of recent H3N2 influenza viruses. PLoS One 7:e Wiley DC, Wilson IA, Skehel JJ Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289: Bush RM, Bender CA, Subbarao K, Cox NJ, Fitch WM Predicting the evolution of human influenza A. Science 286: Skehel JJ, Stevens DJ, Daniels RS, Douglas AR, Knossow M, Wilson IA, Wiley DC A carbohydrate side chain on hemagglutinins of Hong Kong 15

16 influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl Acad Sci U S A 81: Melidou A, Exindari M, Gioula G, Chatzidimitriou D, Pierroutsakos Y, Diza-Mataftsi E Molecular and phylogenetic analysis and vaccine strain match of human influenza A(H3N2) viruses isolated in Northern Greece between 2004 and Virus Res 145: Kitler ME, Gavinio P, Lavanchy D Influenza and the work of the World Health Organization. Vaccine 20 Suppl 2:S WHO. Selection of clinical specimens for virus isolation and of viruses for shipment from National Influenza Centres to WHO Collaborating Centres. Dec _specimens_selected_for_virus_isolation_and_shipment.pdf?ua= Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: Saitou N, Nei M The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: Kimura M A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16: Felsenstein J Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:

17 Network WGIS. Manual for the laboratory diagnosis and virological surveillance of influenza WHO Collaborating Centre MH, London. Report prepared for the WHO annual consultation on the composition of influenza vaccine for the Southern Hemisphere Sep _2.pdf. 19. Pariani E, Amendola A, Ebranati E, Ranghiero A, Lai A, Anselmi G, Zehender G, Zanetti A Genetic drift influenza A(H3N2) virus hemagglutinin (HA) variants originated during the last pandemic turn out to be predominant in the season in Northern Italy. Infect Genet Evol 13: ECDC. Main surveillance developments in weeks Jul 2-15 [cited 2012 Jul 23]. WISO.pdf. 21. Koel BF, Burke DF, Bestebroer TM, van der Vliet S, Zondag GC, Vervaet G, Skepner E, Lewis NS, Spronken MI, Russell CA, Eropkin MY, Hurt AC, Barr IG, de Jong JC, Rimmelzwaan GF, Osterhaus AD, Fouchier RA, Smith DJ Substitutions near the receptor binding site determine major antigenic change during influenza virus evolution. Science 342: Wilson IA, Cox NJ Structural basis of immune recognition of influenza virus hemagglutinin. Annu Rev Immunol 8:

18 Tate MD, Job ER, Deng YM, Gunalan V, Maurer-Stroh S, Reading PC Playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection. Viruses 6: ECDC. Main surveillance developments in week 8/ Feb [cited 2012 Mar 2]. SUR-weekly-influenza-surveillance-overview.pdf. 25. WHO Collaborating Centre for Reference and Research on Influenza National Institute for Medical Research TR, Mill Hill, London, NW7 1AA, UK Report prepared for the WHO annual consultation on the composition of influenza vaccine for the Southern Hemisphere Lin YP, Gregory V, Collins P, Kloess J, Wharton S, Cattle N, Lackenby A, Daniels R, Hay A Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment? J Virol 84: Ampofo WK, Al Busaidy S, Cox NJ, Giovanni M, Hay A, Huang S, Inglis S, Katz J, Mokhtari-Azad T, Peiris M, Savy V, Sawanpanyalert P, Venter M, Waddell AL, Wickramasinghe G, Zhang W, Ziegler T Strengthening the influenza vaccine virus selection and development process: outcome of the 2nd WHO Informal Consultation for Improving Influenza Vaccine Virus Selection held at the Centre International de Conferences (CICG) Geneva, Switzerland, 7 to 9 December Vaccine 31: Schild GC, Oxford JS, de Jong JC, Webster RG Evidence for hostcell selection of influenza virus antigenic variants. Nature 303:

19 WHO. Report prepared for the WHO annual consultation on the composition of influenza vaccine for the Northern Hemisphere 2013/ Feb pdf. 30. WHO. Report prepared for the WHO annual consultation on the composition of influenza vaccine for the Northern Hemisphere 2014/ Feb Sun H, Yang J, Zhang T, Long L, Jia K, Yang G, Webby R, Wan X Using sequence data to infer the antigenicity of influenza virus. MBio 4. Acknowledgements The authors thank Prof. John McCauley, Dr. Rod Daniels, Dr. Yipu Lin, Vicky Gregory and all of their colleagues in the WHO Collaborating Centre for Reference and Research on Influenza at the MRC National Institute for Medical Research, Mill Hill, London for their efforts in testing and analyzing our influenza viruses. 19

20 Figure 1 legend. Weekly distribution of the isolation of the 37 influenza A(H3N2) viruses selected for genetic and antigenic characterization. The dotted line ( ) shows the total number of clinical specimens positive for A(H3N2) (right vertical axis) while the continuous one ( ) indicates the rate of influenza-like illness (ILI) cases of the influenza season (left vertical axis). Specimens tested by the Greek NIC and the WHO CC are shown in black columns while grey bars indicate specimens examined only by the Greek NIC. The numbers of viruses tested are shown on each column. Figure 2 legend. Evolutionary relationships of Greek influenza A(H3N2) strains ( ) with recent European reference and vaccine strains based on the HA1 domain of HA protein gene segment. The evolutionary history was inferred using the Neighbor- Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Only values above 60% are shown. Strains of the influenza season ( ) and influenza season vaccine strains ( ) are depicted. Reference strains are indicated in bold. 20

21 ILI cases/1000 consultations A(H3N2) tested in HPI and WHO-CC A(H3N2) tested in HPI only ILI rate A(H3N2) total number weeks Number of viruses detected

22 A/Athens.GR/76/ A/Athens.GR/52/2012 [WHO CC] 80 A/Iowa/19/ A/Athens.GR/770/ A/Athens/108/2011 A/Perth/10/ A/Thessaloniki/152/2012 A/Ioannina/32/2012 A/Athens.GR/787/2012 A/Athens.GR/748/2012 A/Athens.GR/747/2012 A/Athens.GR/369/2012 A/Athens.GR/237/2012 A/Ioannina.GR/145/2012 [WHO CC] A/Ioannina.GR/81/2012 [WHO CC] 3 A A/Athens.GR/59/2012 [ICU] [WHOO CC] A/Athens.GR/41/ A/Stockholm/18/2011 A/Athens.GR/767/2012 A/Athens.GR/165/2012 [WHO CC] A/Thessaloniki/46/2012 A/Athens.GR/134/2012 [WHO CC] A/Serres/227/ Victoria /208 clade A/Athens.GR/766/2012 A/Thessaloniki/187/2012 A/Thessaloniki/86/2012 [ICU] 74 A/Drama/81/2012 A/Drama/99/2012 A/Thessaloniki/59/ A/Athens.GR/38/2012 A/Athens.GR/112/2012 [WHO CC] A/England/517/2012 A/Athens.GR/69/ B A/Athens.GR/385/2012 [ICU] A/Athens.GR/173/2012 [WHO CC] A/Athens.GR/79/2012 [WHO CC] A/Thessaloniki/105/2012 A/Athens.GR/62/2012 [WHO CC] 77 A/Hong_Kong/3969/2011 A/Victoria/361/2011 A/Athens.GR/769/ A/Athens.GR/16/ C 87 A/Finland/190/2011 A/Norway/1789/2011 A/Athens.GR/131/2012 [WHO CC] A/Athens.GR/208/2012 [WHO CC] A/Serbia/71/ A/Maine/07/ A/Victoria/208/2009 A/Norway/1330/ A/Perth/16/ A/Norway/1186/2011 Perth/16 clade 1 96 A/Beijing-Xicheng/11688/

23 TABLE 1. Amino acid substitutions observed at antigenic sites and N-linked glycosylation sites of HA Amino acid substitutions Glycosylation site Antigenic site C C C C E E E A A A B D D D D D C C C Group Samples A/Perth/16/2009* Q S T D S K K Y K K N L A S T S V P I N E N 3C A/Athens.GR/769/2012 R N I E N - H S A T I K S A/Athens.GR/16/2012 R N I E N - H S A I I H K S (2 gains) A/Athens.GR/208/2012 R N I E N - H S A I I K S A/Athens.GR/131/2012 R N I E N - H S A I I K S A/Finland/190/2011 R N I E N - H S A I I K S A/Athens.GR/62/2012 K E N - S H S A I I S A/Athens.GR/69/2012 E N - S H S A I I S A/Athens.GR/385/2012 E N - S H S A I I S A/Athens.GR/173/2012 E N - S H S A I I S 3B A/Athens.GR/79/2012 E N - S H S A I I S A/Athens.GR/38/2012 R E N - S H S A I I S A/Athens.GR/112/2012 R E N - S H S A I I S (1 gain) A/Thessaloniki/105/2012 E N - S H S A I I S A/Thessaloniki/59/2012 G N - S H S A I I S A/Thessaloniki/187/2012 E N - S H S A I I A/Thessaloniki/86/2012 E N - S H S A I I A/Drama/99/2012 N E N - S H S A I I S A/Drama/81/2012 N E N - S H S A I I A/England/259/2011 A E N - S H S A I I S A/Athens.GR/134/2012 E H - D S H A I I A/Athens.GR/766/2012 E H - D S H A I I

24 3A (1 loss) 6 (1 gain) A/Athens.GR/41/2012 E - D S H A I I A/Athens.GR/59/2012 E - D S H A I I A/Ioannina.GR/81/2012 E - D S H A I I A/Ioannina.GR/145/2012 E - D S H A I I A/Athens.GR/165/2012 E - D S H A I I A/Athens.GR/237/2012 E - D S H A I I A/Athens.GR/369/2012 E - D S H A I I A/Athens.GR/747/2012 E - D S H A I I A/Athens.GR/748/2012 E - D S H A I I A/Athens.GR/767/2012 E R - D S H A I I A/Athens.GR/787/2012 E - D S H A I I A/Thessaloniki/46/2012 E - D S H A I I A/Thessaloniki/152/2012 E - D S H A I I A/Ioannina/32/2012 E - D S H A I I A/Serres/227/2012 E H - D S H A I I A/Stockholm/18/2011 E - D S H A I I A/Athens.GR/52/2012 N E H N - H A A I V A A/Athens.GR/770/2012 N E H N - H A A I L V A A/Athens.GR/76/2012 N E H N - H A A I V A A/Iowa/19/2010 N E H N - H A A I V A * The comparison was performed using the vaccine H3N2 strain for season. Reference strains are indicated in bold The amino acid substitutions are shown using a one-letter amino acid code.

25 TABLE 2. Haemagglutination inhibition (HI) testing results of the Greek strains using the post infection ferret antiserum raised against the egg grown vaccine virus A/Perth/16/2009. Viruses tested Collection date HI titers (Greek NIC) HI titers (WHO CC) Genetic group A/Perth/16/2009* A/Athens GR/79/ B A/Ioannina GR/81/ A A/Ioannina GR/145/ A A/Athens GR/165/ A A/Athens GR/173/ B A/Athens GR/59/ A A/AthensGR/112/ B A/Athens GR/131/ C A/Athens GR/134/ A A/Athens GR/62/ B A/Athens GR/208/ C A/Athens GR/52/ HI testing was performed by both laboratories using 1% guinea pig red blood cells solution; at Greek NIC without oseltamivir and at WHO CC in the presence of

26 oseltamivir. MDCK SIAT-1 cells were used for the propagation of the Greek viruses by both laboratories. *The A/Perth/16/2009 vaccine virus was grown in eggs by the WHO CC and in MDCK SIAT-1 cells by the Greek NIC.