Reserch rticle Muttions cquired during cell culture isoltion my ffect ntigenic chrcteristion of influenz A(H3N2) clde 3C.2 viruses DM Skowronski 1 2, S Siduc 1, C Chmers 1, A Eshghi 3, JB Guy 3 4, M Krjden 1 2, SJ Drews 5 6, C Mrtineu 7, G De Serres 7 8 9, JA Dickinson 10, A Winter 3, N Bstien 11 11 12, Y Li 1. British Columi Centre for Disese Control, Vncouver, Cnd 2. University of British Columi, Vncouver, Cnd 3. Pulic Helth Ontrio, Toronto, Cnd 4. University of Toronto, Toronto, Cnd 5. University of Alert, Edmonton, Cnd 6. Alert Provincil Lortory, Edmonton, Cnd 7. Institut Ntionl de Snté Pulique du Quéec (Ntionl Institute of Helth of Queec), Queec, Cnd 8. Lvl University, Queec, Cnd 9. Centre Hospitlier Universitire de Quéec (University Hospitl Centre of Queec), Queec, Cnd 10. University of Clgry, Clgry, Cnd 11. Ntionl Microiology Lortory, Pulic Helth Agency of Cnd, Winnipeg, Cnd 12. University of Mnito, Winnipeg, Cnd Correspondence: Dnut M. Skowronski (dnut.skowronski@ccdc.c) Cittion style for this rticle: Skowronski D, Siduc S, Chmers C, Eshghi A, Guy J, Krjden M, Drews S, Mrtineu C, De Serres G, Dickinson J, Winter A, Bstien N, Li Y. Muttions cquired during cell culture isoltion my ffect ntigenic chrcteristion of influenz A(H3N2) clde 3C.2 viruses. Euro Surveill. 2016;21(3):pii=30112. DOI: http://dx.doi.org/10.2807/1560-7917.es.2016.21.3.30112 Article sumitted on 10 Decemer 2015 / ccepted on 21 Jnury 2016 / pulished on 21 Jnury 2016 As elsewhere, few (< 15%) sentinel influenz A(H3N2) clde 3C.2 viruses tht dominted in Cnd during the 2014/15 seson could e ntigeniclly chrcterised y hemgglutintion inhiition (HI) ssy. Clde 3C.2 viruses tht could e HI-chrcterised hd cquired genetic muttions during in vitro cell culture isoltion tht modified the potentil glycosyltion motif found in originl ptient specimens nd the consensus sequence of circulting viruses t mino cid positions 158 160 of the hemgglutinin protein. Cution is wrrnted in extrpolting ntigenic reltedness sed on limited HI findings for clde 3C.2 viruses tht continue to circulte glolly. Introduction During the 2014/15 influenz A(H3N2) epidemic, viruses elonging to phylogenetic clde 3C.2 predominted [1,2]. Viruses within this clde ore multiple (10 12) mino cid differences from the A/Texs/50/2012 (clde 3C.1) vccine strin t ntigenic sites of the surfce hemgglutinin (HA) protein [2]. These differences included two clde-defining sustitutions, phenyllnine (F) to tyrosine (Y) sustitution t residue 159 (F159Y) nd lysine (K) to threonine (T) sustitution t the djcent residue 160 (K160T), oth in ntigenic site B [1,2], highly exposed region t the top of the HA protein where muttions crete the potentil for virl evsion of the ntiody response [3,4]. Together with n sprgine (N) residue t position 158, conserved in ll clde 3C viruses, the N158-Y159-T160 sequon in clde 3C.2 viruses represents potentil gin of glycosyltion tht cn msk virl epitopes nd reduce ntiody ccess to the immuno-dominnt ntigenic site B [5,6]. This potentil glycosyltion motif t mino cid positions 158 160 of the HA protein is unique to clde 3C.2 viruses, nd is not found in other recently circulting A(H3N2) genetic cldes. Koel et l. hve previously highlighted positions 158 nd 159 s mong seven residues in the HA protein ssocited with ll mjor ntigenic cluster-trnsition events in A(H3N2) viruses since 1968 [7] nd in recent serologicl nlysis, Chmers et l. highlighted the sustitution t position 159 s likely to hve een responsile for the 2014/15 ntigenic drift [8]. Consistent with these moleculr findings, mid-seson vccine effectiveness (VE) nlyses from multiple countries, including the Cndin Sentinel Prctitioner Surveillnce Network (SPSN), the United Sttes (US) nd the United Kingdom (UK), reported negligile protection ginst the 2014/15 A(H3N2) clde 3C.2 epidemic strin [2,9,10]. In Ferury 2015, the World Helth Orgniztion (WHO) recommended tht the A(H3N2) component for the 2015/16 seson e updted to n A/Switzerlnd/9715293/2013-like (clde 3C.3) strin [11]. Influenz surveillnce reports from reference lortories glolly hve indicted tht circulting A(H3N2) viruses elonging to clde 3C.2 re ntigeniclly 1
Tle 1 Hemgglutintion inhiition ssy titres nd fold reductions reltive to cell- nd egg-pssged A/Switzerlnd/9715293/2013 reference virus for sentinel A(H3N2) virus isoltes with known genetic clde, Cndin Sentinel Prctitioner Surveillnce Network, Novemer 2014 April 2015 (n = 49) Sentinel isolte (clde) n Sentinel isolte HI titre rnge Cell-pssged A/Switzerlnd/9715293/2013 (n = 49) Homologous reference virus HI titre rnge Fold reduction n Sentinel isolte HI titre rnge Egg-pssged A/Switzerlnd/9715293/2013 (n = 35 ) Homologous reference virus HI titre rnge Fold reduction Clde 3C.2 31 80 160 320 640 4 25 160 320 320 640 4 Clde 3C.3 1 160 320 2 1 320 640 2 Clde 3C.3 2 160 640 320 640 4 2 320 320 640 2 Clde 3C.3 15 80 1,280 320 4 7 80 640 320 640 8 HI: hemgglutintion inhiition. These 35/49 viruses were chrcterised in reltion to oth the cell-pssged nd egg-pssged A/Switzerlnd/9715293/2013 reference virus. For the other 14/49 viruses initilly chrcterised in reltion to the cell-pssged A/Switzerlnd/9715293/2013 reference virus, there remined insufficient virl titre to support further chrcteristion in reltion to the egg-pssged reference virus. One clde 3C.3 virus hd n eightfold reduction to egg-pssged A/Switzerlnd/9715293/2013 reference virus; ll other tested viruses hd 4-fold reductions. Tle 2 Potentil glycosyltion motif t hemgglutinin positions 158-160 in influenz A(H3N2) clde 3C.2 viruses from originl ptient specimens nd culture isoltes prior to hemgglutintion inhiition ssy y the NML, Cndin Sentinel Prctitioner Surveillnce Network, Novemer 2014 April 2015 (n = 234) HI ssy chrcteristion Sufficient HA titre for HI ssy (n = 31) Insufficient HA titre for HI ssy (n = 203) N-Y-T n = 219 Originl ptient specimens n = 234 Not N-Y-T n = 2 HA mino cid sequence t positions 158-159-160 Sequence not ville n = 13 N-Y-T n = 156 Not or poly N-Y-T n = 71 Culture isoltes n = 234 Sequence not ville n = 7 26 (12) 1 4 0 (0) 28 (39) 3 193 (88) 1 9 156 (100) 43 (61) 4 HA: hemgglutinin; HI: hemgglutintion inhiition; NML: Ntionl Microiology Lortory; poly: polymorphic for the N-Y-T mino cid sequence (i.e. prtil loss of the potentil glycosyltion motif). Vlues displyed re: numer (% column). A potentil glycosyltion motif is defined y the mino cid sequon: N-X-T/S; where N is sprgine, X is ny mino cid other thn proline nd T/S is either threonine or serine [5,6]. The consensus sequence for clde 3C.2 viruses is N158-Y159-T160, conferring potentil gin of glycosyltion. In the presence of 20 nm oseltmivir. relted to A/Switzerlnd/9715293/2013, despite the fct tht only smll proportion could e chrcterised y conventionl hemgglutintion inhiition (HI) ssy [1,12,13]. Influenz A(H3N2) viruses hve een difficult to chrcterise ntigeniclly y HI ssy due to vrile gglutintion of erythrocytes or loss of ility to gglutinte erythrocytes, prticulr prolem for clde 3C.2 viruses [1]. For the mjority of A(H3N2) viruses tht could not e HI-chrcterised, reference lortories hve imputed ntigenic reltedness sed on sequencing findings, ssuming tht viruses tht could e chrcterised within given genetic group or clde re rodly representtive of circulting strins [12,13]. The Europen Centre for Disese Prevention nd Control (ECDC) hs erlier highlighted tht clde 3C.2 viruses tht hd sufficient HA titre to gglutinte erythrocytes nd tht could e chrcterised y HI ssy hd either lost or were polymorphic for the clde-defining glycosyltion motif t positions 158 160 [14]. To ssess the representtiveness of clde 3C.2 viruses tht could e chrcterised y HI ssy, we exmined mino cid identity t positions 158 160 for the presence of this potentil glycosyltion motif in originl ptient specimens collected y the Cndin SPSN compred with the corresponding sequence fter cell culture isoltion of virus during the 2014/15 seson. 2
Methods Nsl/nsophryngel specimens collected from ptients within seven dys of influenz-like illness onset through the Cndin SPSN etween 1 Novemer 2014 nd 30 April 2015 were tested for influenz y RT-PCR. Influenz-positive specimens were inoculted into Mdin-Dry Cnine Kidney (MDCK), MDCK-SIAT1 or Rhesus Monkey Kidney (RMK) cells to ttempt culture isoltion s per provincil reference lortory protocols. Cell culture isoltes were sumitted to Cnd s Ntionl Microiology Lortory (NML) for ntigenic chrcteristion y stndrd HI ssy protocols using guine pig erythrocytes nd post-infection ferret ntiser supplied y the US Disese Control nd Prevention (US CDC) rised ginst cellnd egg-pssged A/Switzerlnd/9715293/2013 reference viruses [2]. To circumvent ny neurminidse (NA)-medited inding of A(H3N2) viruses to erythrocytes, HI ssys were conducted in the presence of 20 nm oseltmivir croxylte following, where indicted, further single pssge in MDCK-SIAT1 cells t the NML to improve virl titres [15,16]. Antigenic reltedness of sentinel isolte to A/Switzerlnd/9715293/2013 reference virus ws defined s 4-fold reduction in HI ntiody titre compred to the titre of the homologous reference virus [17]. Snger sequencing of the virl HA gene ws conducted on the originl ptient specimens to estlish clde designtion nd to detect mino cid sustitutions in HA ntigenic sites. For the current study, sequencing ws lso conducted on cultured isoltes of clde 3C.2 viruses efore nd fter further pssge in MDCK-SIAT1 cells (if indicted) prior to HI chrcteristion t the NML to ssess mino cid identity reltive to the clde 3C.2 N158-Y159-T160 consensus sequence nd to sequences sed on the corresponding originl ptient specimen. Ethics ords in ech prticipting province pproved the SPSN VE study of which this virologicl su-nlysis is component; virus chrcteristion ws lso conducted s prt of ntionl surveillnce ctivities. Results Clde distriution nd HI chrcteristion Of the 460 influenz A(H3N2) detections y the SPSN during the 2014/15 seson with known clde informtion, 265 (58%) virus isoltes were cultivted y provincil reference lortories nd sumitted to the NML for ntigenic chrcteristion y HI ssy. Of the 265 virus isoltes, 197 (74%) were grown y provincil lortories in MDCK, 44 (17%) in MDCK-SIAT1 nd 24 (9%) in RMK cells. Sumitted A(H3N2) virus isoltes included 234 (88%) viruses elonging to clde 3C.2, 25 (9%) elonging to clde 3C.3, four (2%) elonging to clde 3C.3 nd two (1%) elonging to clde 3C.3, reflecting the overll clde distriution nd clde 3C.2 predominnce mong sentinel A(H3N2) detections previously reported [2]. Of these 265 virus isoltes with known clde informtion, 49 (18%) hd sufficient HA titre to gglutinte erythrocytes nd could e chrcterised y HI ssy. These included only 31 (13%) of the 234 virus isoltes elonging to clde 3C.2. By comprison, of the 31 non-clde 3C.2 virus isoltes, 18 could e chrcterised y HI, including 15 of 25 elonging to clde 3C.3, one of four elonging to 3C.3, nd oth of the virus isoltes elonging to clde 3C.3. All 49 virus isoltes tht could e HI-chrcterised were considered ntigeniclly relted to the cell-pssged A/Switzerlnd/9715293/2013 vccine prototype recommended for the 2015/16 vccine. A suset of 35 of the 49 viruses ws dditionlly chrcterised ginst the egg-pssged A/Switzerlnd/9715293/2013 vccine reference nd 34 of them were considered ntigeniclly relted; one clde 3C.3 virus showing eightfold titre reduction ws considered ntigeniclly distinct (Tle 1). Clde 3C.2 viruses nd the 158 160 sequon Of the 234 clde 3C.2 virus isoltes sumitted to NML, sequencing of the virl HA t positions 158 160 sed on originl ptient specimens ws successful for 221 (94%) viruses (Tle 2). Of these 221 viruses from originl ptient specimens, 219 (99%) ore the clde 3C.2 consensus sequence N158-Y159-T160 consistent with the potentil glycosyltion motif nd two (1%) insted ore K160 found otherwise in clde 3C.2 viruses. Following cell culture isoltion t provincil reference lortories, 229 of 234 (98%) clde 3C.2 virus isoltes hd sequencing informtion ville efore MDCK-SIAT1 pssge (if indicted) t the NML. Of these, 63 (28%) viruses hd lost or prtilly lost (i.e. ecome polymorphic for) the N158-Y159-T160 consensus sequence, including 45 (25%) of 178 grown in MDCK, 10 of 30 grown in MDCK-SIAT1 nd eight of 21 grown in RMK cells. Of the 31 clde 3C.2 viruses tht could e HI-chrcterised, 17 hd undergone single further pssge in MDCK-SIAT1 cells t the NML prior to HI ssy. Sequencing informtion ws ville for 15 of these 17 viruses tht required dditionl MDCK-SIAT1 pssge nd 13 of the 14 viruses tht did not require further MDCK-SIAT1 pssge. Bsed on ville sequencing informtion, ll 28 viruses hd lost or were polymorphic for the potentil glycosyltion motif (Tle 2). For ll ut two of the virus isoltes tht were further pssged in MDCK-SIAT1 cells t the NML, sequences were identicl efore nd fter tht pssge. Of the two viruses modified with MDCK-SIAT1 pssge t the NML, one isolte tht ws polymorphic fter initil cell culture lost the potentil glycosyltion motif nd one isolte tht mintined the originl consensus sequence fter initil cell culture ecme polymorphic fter MDCK-SIAT1 pssge. 3
Tle 3 Amino cid sequence t hemgglutinin positions 158 160 of influenz A(H3N2) clde 3C.2 viruses with respect to the potentil glycosyltion motif in finl culture isoltes prior to hemgglutintion inhiition ssy y the NML, Cndin Sentinel Prctitioner Surveillnce Network, Novemer 2014 April 2015 (n = 234) HA mino cid sequence 158 159 160 Glycosyltion motif Frequency n (%) Interprettion Consensus sequence in circulting clde 3C.2 viruses N Y T + CHO Sequence fter finl cell culture A(H3N2) clde 3C.2 viruses with sufficient HA titre for HI ssy (n = 31) N Y K CHO 8 Reversion to clde 3C.2 K160 N Y A CHO 7 New T160A muttion N Y T/K Polymorphic c 4 Prtil reversion to clde 3C.2 K160 K Y T CHO 3 New N158K muttion N Y I CHO 3 New T160I muttion D Y T CHO d 1 New N158D muttion H Y T CHO 1 New N158H muttion N/S Y T Polymorphic 1 Polymorphic for new N158S muttion Sequence not ville NA 3 NA A(H3N2) clde 3C.2 viruses with insufficient HA titre for HI ssy (n = 203) N Y T + CHO 156 (77) Consensus clde 3C.2 sequence N/K Y T Polymorphic 6 (3) Polymorphic for new N158K muttion N/D Y T Polymorphic 5 (2) Polymorphic for new N158D muttion N Y T/I Polymorphic 5 (2) Polymorphic for new T160I muttion N Y T/A Polymorphic 5 (2) Polymorphic for new T160A muttion N Y T/K Polymorphic 5 (2) Prtil reversion to clde 3C.2 K160 N Y A CHO 3 (1) New T160A muttion N Y I CHO 3 (1) New T160I muttion N Y K CHO 3 (1) Reversion to clde 3C.2 K160 S Y T CHO 2 (1) New N158S muttion D Y T CHO 1 (0) New N158D muttion K Y T CHO 1 (0) New N158K muttion N/K Y T/I Polymorphic 1 (0) Polymorphic for new N158K nd T160I muttions N/R Y T/I Polymorphic 1 (0) Polymorphic for new N158R nd T160I muttions N/S Y T/A Polymorphic 1 (0) Polymorphic for new N158S nd T160A muttions N/K/R/S Y T Polymorphic 1 (0) Polymorphic for new N158K/R/S muttion Sequence not ville NA 4 (2) NA CHO: cron-hydrogen-oxygen (i.e. glycosyltion); HA: hemgglutinin; HI: hemgglutintion inhiition; NA: not ville; NML: Ntionl Microiology Lortory. + CHO: potentil glycosyltion motif in clde 3C.2 viruses defined y the mino cid sequon: N158-Y159-T160 [5,6]; CHO: loss of this potentil glycosyltion motif; Polymorphic: prtil loss of the glycosyltion motif. Muttions t residues 158 160 compred with the consensus sequence for clde 3C.2 viruses re shded in lue: drk lue shding indictes mino cid muttion reltive to the consensus sequence; light lue shding indictes polymorphism reltive to the consensus sequence. Two viruses were N158-Y159-K160 ( CHO) in the originl ptient specimen, including one tht could nd one tht could not e HI-chrcterised. Finl ville sequence of virus isoltes prior to HI chrcteristion is shown. Of the 234 viruses sent to the NML, 220 were re-pssged in MDCK-SIAT1 cells to ttempt to improve virus titre, including 17 of 31 tht could e HI-chrcterised nd ll 203 tht could not e HIchrcterised. Sequences for cell culture isoltes s sumitted from provincil reference lortories re shown for the 13 of 14 viruses with ville sequence informtion tht could e HI-chrcterised without further pssge in MDCK-SIAT1 cells t the NML. In the presence of 20 nm oseltmivir. c One of these four viruses ws T160 (i.e. + CHO) in the cell culture isolte efore further MDCK-SIAT1 pssge t the NML ut ecme polymorphic fter MDCK-SIAT1-pssge with prtil reversion to T/K160 (i.e. N158-Y159-T/K160). d This virus ws polymorphic for the glycosyltion motif with N/D158 in the cell culture isolte efore MDCK-SIAT1 pssge t the NML ut lost the potentil glycosyltion motif (i.e. ecme CHO) fter MDCK-SIAT1 pssge with D158 (i.e. D158-Y159-T160). 4
Tle 4 Reference hemgglutinin sequences from the GISAID EpiFlu dtse used to ssess the 158 160 sequon in the southern hemisphere 2016 influenz A(H3N2) cell- nd egg-pssged vccine reference strin (clde 3C.2) Segment ID Collection dte Isolte nme Originting lortory Sumitting lortory Authors Pssge history 158 159 160 sequon EPI539576 26 Fe 2014 EPI578430 1 Jn 2014 EPI643118 26 Fe 2014 EPI614406 1 Jn 2014 EPI614421 1 Jn 2014 EPI614414 1 Jn 2014 X-263 X-263A X-263B Government Virus Unit Crick Worldwide Influenz Centre Crick Worldwide Influenz Centre New York Medicl College New York Medicl College New York Medicl College Ntionl Institute for Medicl Reserch Disese Control nd Prevention Ntionl Institute of Infectious Diseses (NIID) Disese Control nd Prevention Disese Control nd Prevention Disese Control nd Prevention Tkshit, Emi; Fujiski, Seiichiro; Shirkur, Msyuki; Wtne, Shinji; Odgiri, Tkto MDCK-SIAT1 E5/E1 E6(Am1AI)/E1 + 1 EX EX EX N-Y-T GISAID: Glol Inititive on Shring All Influenz Dt. Originting country for ll isoltes displyed is Hong Kong Specil Administrtive Region (SAR). The consensus sequence for clde 3C.2 viruses is N158-Y159-T160, shown s N-Y-T for the cell (MDCK-SIAT1)-pssged (clde 3C.2) reference virus (first row in Tle) nd conferring potentil gin of glycosyltion. In the egg-pssged reference (clde 3C.2) reference viruses (rows 2 6 in Tle), the potentil glycosyltion motif is lost due to reversion to clde 3C.2 K160. Of the 203 clde 3C.2 viruses tht could not e HI-chrcterised, ll hd undergone single further pssge in MDCK-SIAT1 cells t the NML to improve virl titre. Before MDCK-SIAT1 pssge, 165 of the 199 (83%) virus isoltes with ville sequencing informtion pre-mdck-siat1 pssge hd the potentil glycosyltion motif ssocited with the clde 3C.2 consensus sequence N158-Y159-T160. After pssge in MDCK-SIAT1 cells, 156 (78%) of the 199 viruses tht hd sequence informtion ville post-mdck-siat1 pssge hd the potentil glycosyltion motif (Tle 2). Among viruses with ville sequencing informtion (n=227), the sence of the potentil glycosyltion motif t positions 158 160 in the finl virus isolte ws significntly ssocited with the ility to HI-chrcterise viruses (0 with the glycosyltion motif mong 28 tht could e HI-chrcterised vs. 156 (78%) with the glycosyltion motif mong 199 tht could not e HI-chrcterised; chi-squre = 70.2, degrees of freedom = 1, p-vlue < 0.001). Specific muttions t positions 158 nd 160 of finl virus isoltes influencing the potentil glycosyltion motif prior to HI chrcteristion re shown in Tle 3; the F159Y clde mrker for 3C.2 viruses ws conserved in ll isoltes. Discussion Similr to reports elsewhere, only smll proportion (< 15%) of clde 3C.2 viruses collected through the Cndin SPSN during the 2014/15 seson were le to gglutinte guine pig erythrocytes for ntigenic chrcteristion y HI ssy [1,12,13]. All clde 3C.2 virus isoltes tht could e chrcterised were considered ntigeniclly relted to the 2015/16 vccine strin, lthough more vriility in HI results, prticulrly in reltion to the egg-pssged reference virus, hs een reported y other surveillnce systems [1]. Our findings, however, suggest tht the smll proportion of clde 3C.2 viruses tht could e chrcterised y HI ssy were not representtive of circulting viruses with respect to the clde-defining potentil glycosyltion motif t positions 158 160. We show tht cell culture pssge, whether in MDCK, MDCK-SIAT1 or RMK cells, tht is required for HI chrcteristion, cn fully or prtilly lter the N158-Y159-T160 sequon. This sequon in circulting clde 3C.2 viruses is ssocited with predicted gin of glycosyltion tht my e relevnt for ntiody inding [6]. While these findings corroorte n erlier report of this effect y the ECDC [14], here we provide direct quntifiction nd comprison of virl genomic sequences in originl ptient specimens compred with culture isoltes, highlighting loss of the potentil glycosyltion 5
motif s n rtefct of in vitro cell culture isoltion. This type of ssessment hs not een widely reported elsewhere, in prt ecuse most virl sequences, including those posted to pulic dtses, re sed on culture isoltes nd re not directly compred with primry specimens. However, understnding how virl culture impcts genetic identity efore ntigenic chrcteristion is criticl to interpreting nd extrpolting reltedness mong vccine nd circulting strins, for the purpose of nticipting vccine performnce nd for vccine strin selection. Limittions of our nlysis include the well-recognised vriility in the HI ssy [17], nd the smll numer of viruses tht could e ntigeniclly chrcterised for sequence comprison. Antigenic chrcteristion of greter numer of A(H3N2) viruses, prticulrly those elonging to clde 3C.2, my e possile through use of ssys tht do not rely on gglutintion of erythrocytes, such s neutrlistion ssys [14,18]. The ECDC nd others hve considered the N158-Y159-T160 sequon to e potentil gin of glycosyltion in the mjority of clde 3C.2 viruses [1,5,6]; the glycosyltion potentil of this motif sed on the clde 3C.2 consensus sequence is 0.65 using NetNGlyc 1.0, where the threshold for glycosyltion potentil is 0.5 [19]. To further delinete the N-glycosyltion effect of the K160T muttion, in vitro studies should e done to specificlly ssess the interply etween this muttion, its resulting glycosyltion potentil nd ntiody inding t ntigenic sites. We show sttisticlly significnt effects of the N158-Y159-T160 sequon on the ility to chrcterise viruses y HI ssy, ut it is uncler how much this potentil glycosyltion motif contriutes to chllenges in ntigenic chrcteristion using ntiody titrtion ssys. A proportion of clde 3C.2 viruses (43/199; 22% in this nlysis, Tle 2), s well s other A(H3N2) genetic sugroups, tht lck or re polymorphic for this glycosyltion motif hve lso een difficult to ntigeniclly chrcterise y HI ssy, suggesting tht other fctors, such s virl lod nd vidity to silic cid receptors, re lso likely to contriute [20]. Our gol, however, ws not to investigte those fctors ut to ssess the representtiveness of clde 3C.2 viruses tht could e chrcterised y HI ssy in reltion to the mjority tht could not e chrcterised, with respect to the potentil glycosyltion motif t pivotl ntiody inding positions 158 160 of ntigenic site B. Our findings suggest tht cution is wrrnted in extrpolting ntigenic reltedness sed on limited HI results for A(H3N2) clde 3C.2 viruses. Clde 3C.2 viruses hve continued to predominte mong A(H3N2) detections throughout the 2015 southern hemisphere influenz seson nd erly into the 2015/16 northern hemisphere seson [1,18]. Despite vccine reformultion, clde 3C.2 viruses still differ from the northern hemisphere 2015/16 clde 3C.3 vccine strin y 10 12 mino cids t ntigenic sites, including the sme N158-Y159-T160 glycosyltion motif tht distinguished them from the 2014/15 vccine [2]. For the southern hemisphere s 2016 influenz seson, the WHO hs recommended chnge to n (H3N2)-like (clde 3C.2) representtive vccine virus [18], for which the egg-pssged reference strin ers K160 rther thn T160 nd thus lso seems to hve lost the potentil glycosyltion motif (Tle 4). Clrifying the significnce of the N158- Y159-T160 potentil glycosyltion motif in circulting clde 3C.2 strins thus remins criticl to the interprettion of ntigenic reltedness nd to expecttions of vccine-induced ntiody protection, for which ongoing epidemiologicl monitoring of VE will e importnt. Acknowledgements Funding ws provided y the Cndin Institutes of Helth Reserch (CIHR grnt # TPA-90193), the British Columi Centre for Disese Control, Alert Helth nd Wellness, Pulic Helth Ontrio, Ministère de l snté et des services sociux du Quéec nd the Institut ntionl de snté pulique du Quéec nd the Pulic Helth Agency of Cnd. The uthors grtefully cknowledge the contriution of sentinel sites whose regulr sumission of specimens nd dt provide the sis of our nlyses. We wish to cknowledge the coordintion nd technicl support provided y epidemiologicl nd lortory stff in ll prticipting provinces. We thnk those who provided lortory support t the British Columi Pulic Helth Microiology nd Reference Lortory, the Alert Provincil Lortory, the Pulic Helth Ontrio Lortory nd the Lortoire de snté pulique du Quéec. Conflict of interest Reserch grnts for unrelted studies: GDS - GlxoSmithKline (GSK); JG - Pfizer; MK - Roche, Merck, Gen-Proe nd Siemens. 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