WHO INFLUENZA CENTRE LONDON. Report

Transcription

1 WHO INFLUENZA CENTRE LONDON Report September 009

2 WHO Collaborating Centre for Reference and Research on Influenza National Institute for Medical Research The Ridgeway Mill Hill London, NW7 AA Dr. Alan J. Hay (Director) Dr. Rodney Daniels (Deputy Director) Dr. Yi Pu Lin (Assistant Director) Dr. Xiang Zheng Dr. Ting Hou Ms Victoria Gregory Ms Lynne Whittaker Mr Johannes Kloess Mr Nicholas Cattle Tel: ; Fax: ahay@nimr.mrc.ac.uk; whocc@nimr.mrc.ac.uk Acknowledgements We thank all who have contributed information and viruses, and associated data, to the WHO Global Influenza Surveillance Network, which provide the basis for our current understanding of recently circulating influenza viruses, and this brief summary. In addition to data shared among the other WHO Collaborating Centres, sequence data for viruses isolated during this period were received from Influenza Centres in Austria, Denmark, Finland, France, Germany, Hong Kong, Italy, Netherlands, Norway, Portugal, South Africa, Sweden, Switzerland and UK. Page of

3 Influenza activity, February to September 009 Influenza during this period was dominated by the emergence of the pandemic A(HN) virus. Although infections in many countries continued to be due largely to imported cases (travellers), the virus has predominated in most of the countries where it has circulated more widely. Seasonal influenza A(HN), A(HN) and B viruses have continued to circulate at low levels. Thirty six cases of H5N infection were reported since the beginning of February, including four fatal cases in Vietnam during February to April and cases in Egypt, four of which were fatal. During February and March in the northern hemisphere, influenza continued to be relatively mild. HN viruses predominated in Europe, where the season was effectively over by mid April (week 6), although influenza B rose to prominence late in the season, accounting for 6% of viruses in total, the majority (97%) of which were of the B/Victoria//87 lineage (Figure ). In contrast, circulation of HN viruses was relatively low in North America where HN (seasonal) predominated in the USA and influenza B was prevalent together with HN viruses in Canada, as they were in Japan. HN, HN and B viruses co-circulated in many northern hemisphere and tropical countries in Africa and Asia. Increased activity in some Asian countries from June to August included outbreaks of HN in China. Following detection of the pandemic A(HN) virus in late April, this virus, which had been responsible for the unseasonal outbreaks of influenza in Mexico during February and March, steadily increased in prevalence in North America, peaking during June in the USA, and spreading to other parts of the world. In Europe, the virus was initially detected in late April (week 8) and peak detections, sentinel and non-sentinel, were recorded in the second half of July (weeks 9-) (Figures and ). As of th September, ECDC reported 50,778 confirmed cases with 6 deaths (Figure d). The UK experienced the highest incidence of pandemic HN infection, with, confirmed cases recorded, as of 9 th September, with 76 associated deaths (Figure c). All four UK national sentinel influenza schemes showed unseasonal rises in ILI consultation rates, peaking in late July, with levels in England and Wales being higher than those for the 008/09 season (Figure a), although not exceeding the epidemic activity threshold (Figure b). The proportion of specimens testing positive for pandemic HN reached 0%, compared to 60% positive for influenza at the peak of the 008/09 season (Figure d). Few seasonal influenza viruses were detected during circulation of the pandemic HN virus between June and September (Figure d). From August, marked increases in the prevalence of the pandemic HN virus occurred in many African and Asian countries, including for example China, Japan and Kenya. During the winter season in the southern hemisphere, influenza was due mainly to the pandemic HN virus where it was prevalent in South American countries, Australia and New Zealand during June and July, and largely replaced previously circulating seasonal HN and HN viruses. Pandemic HN virus emerged somewhat later in South Africa during August, following widespread outbreaks due to HN in June.

4 Antigenic and genetic characteristics of pandemic A(HN) viruses and seasonal A(HN), A(HN) and B viruses isolated during February to August 009 Of approximately 00 viruses isolated during February to April, the majority (5%) were A(HN) and 0% were A(HN), with influenza B accounting for 5% of the viruses characterized (Table ). Following detection of the pandemic HN virus at the end of April, it has been predominant among clinical samples and 8 viruses isolated from May to August. During the latter period, HN viruses accounted for %, while the proportions of seasonal HN (5%) and B (0%) viruses also decreased (Table ). B/Victoria lineage viruses accounted for the majority (88%) of B viruses isolated from Feb to July. Pandemic A(HN) viruses With few exceptions, HI tests showed that the pandemic HN viruses continued to be antigenically similar to the prototype vaccine virus A/California/7/009 (Table ). Many of the HA and NA sequences of recent isolates possess the common changes S0T in HA and V06I and N8D in NA, relative to the vaccine virus (Figures and 5). While all viruses tested were resistant to amantadine/rimantadine, sporadic detections of oseltamivir resistance, due to the H75Y mutation (Figure 5), have been reported; all such viruses retain sensitivity to zanamivir. Seasonal A(HN) viruses Seasonal HN viruses continued to circulate at low levels. In HI tests, most were shown to be antigenically closely related to recent A/Brisbane/59/007-like reference viruses, such as A/Seychelles/9/008 (clade B) or A/Hong Kong/856/008 (clade C), although HI titres were generally reduced, relative to homologous, with the antiserum to egg-grown A/Brisbane/59/007 (Table ). HAs of clades B and C have, in general, been indistinguishable in HI tests. The HA and NA sequences of the majority of recent isolates fell within clade B and variation in amino acid sequence was similar to that observed earlier in 009 (Figures 6 and 7). Different subgroups of HA were distinguished by the amino acid changes SN and G85A, N8S and G85S, or H9R/N, but were not distinguishable in HI tests (Table ). The majority of viruses (clade B) tested were resistant to oseltamivir, retaining the H75Y mutation in NA (Figure 7), and were sensitive to zanamivir and amantadine/rimantadine.

5 A(HN) viruses Most A(HN) viruses gave higher HA titres with guinea pig RBCs than with turkey RBCs and many failed to agglutinate the turkey cells. Most recent HI tests therefore used guinea pig RBCs. HI tests with the viruses which did not agglutinate turkey RBCs showed that many were antigenically closely related to the vaccine viruses A/Brisbane/0/007 and A/Uruguay/76/007, whereas others were antigenically distinct and closely related to recent reference viruses, such as A/Hong/Kong/985/009 and A/Perth/6/009 (Table 5). As previously observed, most of the viruses that agglutinated both turkey and guinea pig RBCs gave reduced HI titres, relative to homologous, as shown for the MDCK cell-grown reference viruses A/Trieste/5c/007, A/Finland/9/008 and A/Johannesburg/5/008 in Table 5. Oseltamivir was observed to reduce (or abolish) haemagglutination by many MDCK cellgrown viruses, especially of turkey RBCs, and it was apparent that attachment by the NA contributed to haemagglutination. Studies of reverse genetics derived viruses have recently shown that this is due to the presence of a D5G mutation causing irreversible binding to the RBCs. Addition of oseltamivir (0 nm) to HI tests with guinea pig RBCs abolished NA-dependent agglutination and allowed analysis of haemagglutination inhibition due specifically to anti-ha antibody and a clearer antigenic distinction between Brisbane/0/007-like viruses and the recently emergent antigenic variant (Tables 6 and 7). Virus neutralization tests also showed that A/Hong/Kong/985/009 was antigenically distinct from A/Brisbane/0/007-like viruses (Table 8). Overall, many of the viruses were shown to be antigenically and genetically closely related to A/Brisbane/0/007 and A/Uruguay/76/007 vaccine strains, while an increasing proportion of recent isolates were antigenically and genetically closely related to the emergent antigenic variant, represented by A/Hawaii/7/009, A/Hong Kong/985/009 and A/Perth/6/009 reference viruses. The HA sequences of the latter viruses formed a separate clade characterized by the amino acid substitutions E6K, NK, K58N and N89K (plus VA, or I60M and R6Q) (Figure 8). The locations of these substitutions on the surface of the HA molecule is illustrated in Figure 9. There were no corresponding distinctive amino acid changes in the NA sequences of these viruses that distinguished them from the A/Brisbane/0/007-like viruses (Figure 0). Viruses distinguished by the substitutions K58N, N89K and TA in HA were also antigenically closely related to A/Hong Kong/985/09 (Table 6); their NAs were characterized by the changes I6T and Y0C in the stalk of the molecule. Several of the recent A/Brisbane/0/007-like viruses had acquired other changes in HA, K58R plus VI, or P0S, A8S and T8N, and in NA, S67N and K69T. All the viruses tested possessed asparagine in M, which confers resistance to amantadine and rimantadine. No resistance to NA inhibitors was detected.

6 B viruses Influenza B viruses were predominantly of the B/Victoria-lineage. The majority of B/Victoria-lineage viruses were antigenically closely related to the vaccine strain B/Brisbane/60/008, although viruses from Hong Kong and Singapore, in particular, were closely related to the reference virus B/Hong Kong/5/05 (B/Malaysia/506/0-like) (Table 9). HA sequences of most of the former fell within the Brisbane/60 clade and were similar to those of earlier B/Brisbane/60/008-like viruses, whereas those of the latter viruses were closer to the sequences of earlier B/Malaysia/506/00-like viruses and Chinese isolates, such as B/Jiangsu-Gulou/5/009 or B/Hubei-Songzi/5/008, which lack the N65K substitution (Figure ). As previously, the NA sequences fell within corresponding clades and subclades of the B/Victoria-lineage (Figure ). The few B/Yamagata-lineage viruses were shown to be antigenically closely related to the previous B/Brisbane//007 vaccine strain and/or more recent reference viruses, such as B/Bangladesh//007 or B/Valladolid/8/008 (Table 0). Most of their HA and NA sequences belonged to the Bangladesh//07 clade (Figures and ). A(H5N) viruses No human isolates were received. Sequences of the 8 genes of two chicken viruses from Egypt, isolated during February, showed that they were similar to those of other contemporary avian and human clade. isolates, as shown for HA and NA in Figures and 5. The HAs of these recent Egyptian isolates were characterized by the amino acid substitutions DN, S0N, I5T, P5S and S57F and a deletion of residue 9. The NAs were characterized by the substitutions LM and S50G (plus additional substitutions RK or QK and VI, or P0S and V89G). No drug resistance mutations were detected in the NA or M proteins.

7 Figure : Influenza positive specimens 008/9 season - Europe. Snapshot taken at week 6/009. Season effectively over by week6/009. Predominantly an A(HN) season. Pandemic HN picked up initially in nonsentinel sampling. Data taken from EuroFlu.org Page 7 of

8 Figure : Pandemic (HN) 009 virus detections Europe (ECDC data). a) Number of sentinel specimens positive for influenza by type, subtype and week of report. b) Proportion of sentinel samples positive for influenza by week of report. c) Number of non-sentinel specimens positive for influenza by type, subtype and week of report. d) Number of confirmed deaths among pandemic (HN) 009 influenza cases by week of notification: EU and EFTA countries. Page 8 of As of.09.09: confirmed cases, 6 deaths.

9 Figure : HPA (UK) National Influenza Report, (week 7). a) GP weekly consultation rates for influenza/ili in the UK national sentinel influenza schemes, 008/09. c) Cumulative number of laboratory confirmed cases, as of * * 76 deaths. d) Number of samples testing positive for influenza in the two GP-based English sentinel virological schemes. b) RCGP weekly consultation rate for ILI 008/09 and recent years. Page 9 of

10 Table. Geographic origins of human influenza isolates characterised, February August 009 HN HN B HN HN B Country A/Bris A/Cal A/Bris A/HK Yamagata Victoria Country A/Bris A/Cal A/Bris A/HK Yamagata Victoria 59/07 7/09 0/07 985/09 lineage lineage 59/07 7/09 0/07 985/09 lineage lineage Fl//06 Bris/60 HK/5/05 Fl//06 Bris/60 HK/5/05 February 009 May 009 Albania Belgium 7 Belgium 5 Finland Cameroon France Croatia Germany Czech Republic Georgia Egypt Ghana Estonia Greece France 0 Hong Kong Georgia 5 Iran Ghana Ireland 7 Greece Israel Iran Italy Italy 5 Luxembourg Latvia 5 Madagascar 5 Luxembourg Madagascar Netherlands Norway 5 Russia Sweden Serbia 7 South Africa 5 Spain Ukraine Sweden 5 United Arab Emirates Switzerland United Kingdom 6 Ukraine 5 June 009 March 009 Algeria Croatia Austria Egypt Belgium Estonia Bulgaria Finland Cameroon Georgia 7 Cyprus 6 Hong Kong Denmark Iran Estonia Luxembourg France Madagascar Ghana 8 Moldova Hong Kong Norway Ireland Russia 5 Italy 6 Serbia 5 Latvia Singapore Luxembourg Sweden Macedonia Switzerland Madagascar 9 Ukraine 7 Malta April 009 Mauritius Croatia Morocco Egypt Netherlands Finland Norway 8 France Portugal 8 Georgia Singapore Ghana Senegal Hong Kong Serbia and Montenegro Madagascar 5 Singapore 5 Netherlands Slovenia Norway South Africa 0 Russia Sweden Singapore United Arab Emirates Spain Qatar 5 South Africa July 009 Ukraine 6 Austria Bulgaria 9 Cameroon 0 Subtotal = Ghana 0% % 58 (5%) 0 (5%) Greece Hong Kong Italy Latvia 5 Macedonia Malta 6 Mauritius Moldova Portugal 5 Slovenia United Kingdom August 009 Cameroon Luxembourg Mauritius Norway Romania Singapore Spain 7 5 Subtotal = % 6% 0 (%) (0%) Page 0 of

11 Table. Summary of human influenza isolates characterised, February August 009 HN HN Month of A/Bris A/Cal A/Bris A/HK Yamagata Collection 59/07 7/09 0/07 985/09 lineage B Victoria lineage Fl//06 Bris/60 HK/5/05 February March April May June July August Total = % 7% 59 (6%) (5%) Page of

12 Table. Antigenic analyses of pandemic influenza A(HN) viruses Haemagglutination inhibition titre Post infection ferret sera Viruses Collection Passage A/NJ A/sw/Iowa A/NJ A/Cal A/Cal A/Eng A/Auck A/Narita A/Ohio A/Bris date History 8/76 5/0 8/76 /09 7/09 95/09 /09 /09 /07 59/07 R5/79 F/99 F/8 C/F/09 C/6/09 NIBSC F7/09 C/7/09 JAP 6/09 CDC 97/08 F5/08 REFERENCE VIRUSES A/Iowa/5/0 Ex < < < < < < < < A/New Jersey/8/76 Ex > < A/Ohio//007 C / MDCK 560 < < A/California//009 C,M,E < A/California/7/009 E+ 560 < < A/England/95/009 MDCK 560 < < A/Auckland//009 Ex < A/Narita//009 E < A/Brisbane/59/007 E7 < < < < < < < < < 0 TEST VIRUSES A/Osaka/80/009 Cx /MDCK 560 < < A/Brandenburg/9/009 0/05/009 Siat,MDCK 560 < < < A/CastillayLeon//009 /05/009 MDCK/MDCK 80 < ND < A/Lyon/0/009 8/05/009 MDCK+ 560 < < A/Ukraine/ 59/009 9/05/009 MDCK 560 < ND < A/Belgium/ 8/009 /05/009 MDCK 560 < ND < A/Abu Dhabi/78orig/009 Jun-09 MDCK 50 < >50 50 < A/Cyprus/S/009 Jun-09 MDCK 560 < < A/Ireland/5770/009 Jun-09 MDCK 80 < ND < A/Macedonia/6/009 Jun-09 MDCK 80 < < A/Slovenia//009 Jun-09 MDCK 560 < < A/Norway/7/009 08/06/009 MDCK+ 50 < > < A/Denmark/58/09 Res 09/06/009 sw kidney, MDCK 560 < ND < A/Qatar/89/009 7/06/009 MDCK 80 < < A/Singapore/99/009 7/06/009 MDCK < < A/Dakar/0/09 8/06/009 MDCK 560 < < A/Montenegro/86/009 /06/009 MDCK 560 < > < A/Luxembourg/87/009 /06/009 MDCK+ 560 < >50 50 < A/Marrakech/7/009 /06/009 MDCK 80 < < A/Estonia/8FA/009 /06/009 MDCK 80 < < A/South Africa//009 /06/009 MDCK 80 < < A/Norway/00/009 6/06/009 MDCK/MDCK 560 < < A/Algeria/G65/009 8/06/009 MDCK 80 < < A/Greece/66/009 Jul-09 MDCK 560 < < A/Malta/MV5665/009 Jul-09 MDCK 560 < < A/Lisboa/9/009 0/07/009 MDCK/MDCK 560 < < A/Austria/508/009 06/07/009 C / MDCK 60 < < ND ND < A/Italy/case87/09 07/07/009 MDCK 560 < < A/Latvia/7-655/009 /07/009 MDCKx/MDCK 560 < < A/Austria/50995/009 /07/009 MDCK+ 560 < ND < A/Hong Kong/558/009 8/07/009 MDCK+ 50 < < A/Bulgaria/0/009 /07/009 MDCK 80 < ND < A/Modra/56/009 7/07/009 MDCK 80 < ND < A/Moldova9/009 /07/009 MDCK 560 < < A/Iran/5/009 Aug-09 E / E 560 < ND ND < A/Bratislava/58/009 09/08/009 MDCK 560 < ND < A/MauritiusAA/009 06/08/009 MDCK 560 < ND <. < = 0;. hyperimmune rabbit serum Page of

13 Figure. Phylogenetic comparison of pandemic influenza A HN HA genes Vaccine strains Isolates Apr -May 009 June - Jul 009 Aug 009. Oseltamivir resistant LI Q9H S8Q A97T VI 6AA differences DE S0T hbratislava5809l hmoldova09 hsouthafrica009 hmauritiuskp0909ha hmauritiusfj0909fahag hcypruss09ha hdenmark5809ha. hdenmark509mha hportugalgs909csha hestoniabtha hqatar8909ha hukraine5909csha hthessaloniki09hal hromania09ha hsouthafrica0609 hthessaloniki6609 hohio0709 halgeriag609u hengland009eha hireland59709ha hluxembourg80609ha hmontenegro8609ha hmalta hsingapore009u hestonia809fau hsingapore5709cdcy. hyamaguchi09japj. hmalta hmoscowiiv009dbl halgeriag6609ha hbulgaria009hal hmacedonia609 hmodra5609l hbelgium8609ha habudhabiis09ha hrabat709l hathens7609ha hbratislava5809hag hmacedonia709ss hireland59609ha hnorway6809ha hnorway7709ha hmarrakech709u hslovenia68709ha hisrael7609ha hsingapore5809u hluxembourg8709ha hdakar09hau hlatvia609mha hiceland09ha hportugal09a hbrandenburg909hay hkansas009 hcatalonia509ha hcalifornia709dba hcalifornia09cdc hnetherlands6009db htexas509dba. hmexico86009 habudhabi09ha hhunanswl09u hmaltapsha haustria500009hal hslovenia0609ha hbelgium909ha hosaka8009japj. salbertaoth809dbj habudhabiha hisrael609a sguangxi705db sindianap900db sohio5507db skansas0060db hohio55988db swisconsin897db hiowa06ha skoreacas0805db sontario0db hnewjersey876db 0.0 Page of

14 Figure 5. Phylogenetic comparison of pandemic influenza A HN NA genes Vaccine strains Isolates Apr -May 009 June - Jul 009 Aug 009. Oseltamivir resistant AA differences H75Y hmauritiuskp09n hmauritiusfj09ng hsouthafrica009na hcypruss09n hbratislava5809nal hmoldova09nal hsingapore5709ncdcy. hdenmark5809n. hosaka8009njapj. hyamaguchi09njapj. hhunanswl09nu. hportugalgs909csn hestoniabtn hestonia09na hireland59709n hireland59609n hthessaloniki6609nal hbratislava5809ng hqatar8909na halgeriag6609na hbelgium8609n hluxembourg80609na hdenmark509mn habudhabiis09na hiceland09n hengland009en hukraine5909csn hathens7609na hthessaloniki09nal hdakar09nu hsingapore009nau hmalta567709na hlativia609mn hisrael7609na hmoscowiiv009ndbl hrabat709nal hmontenegro8609na hmodra5609nal hsingapore5809nau hmalta na halgeriag609nau hbulgaria009nl hmacedonia6na hsouthfrica0609na hromania09na hmacedonia7na hportugal09n hcatalonia509n hbrandenburg909ny hcalifornia709db hcalifornia09cdcn hslovenia0609na hluxembourg809n hmarrakech709nau htexas509dbna hnorway6809n hnorway7709n hthessaloniki809nal hbelgium909na habudhabin habudhabi09n hslovenia68709na hisrael609n hnetherlands6009ntn salbertaoth809ndbj hmaltapsna haustria500009nl sswitz7905n sit509697nagb sit95nagb sit598n sfin8998n sca680na V06I N8D nswitz Page of

15 Table. Antigenic analyses of seasonal influenza A HN viruses Viruses Collection Passage A/NC A/HK A/SI A/Neth A/Bris A/SP A/Sey A/HK A/HK A/Mos A/HK Date History 0/99 65/06 /06 5/07 59/07 /08 9/08 856/08 870/08 /09 988/09 F9/08 F/06 F8/07 F/07 F6/08 F7/08 F7/08 F8/08 F/09 F9/09 F0/09 Phylogenetic Clade C A B B B B C C B B REFERENCE VIRUSES A/New Caledonia/0/99 06/09/999 Ex A/Hong Kong/65/006 7/07/006 E E A/Solomon Islands//006 /08/006 E E A/Netherlands/5/007 /0/007 MDCK Siat A/Brisbane/59/007 0/07/007 E E A/St.Petersburg//008 /0/008 E E < A/Seychelles/9/008 0/06/009 MDCK Siat < A/Hong Kong/856/008 /07/008 MDCK Siat A/Hong Kong/870/008 6/07/008 MDCK Siat < A/Moscow//009 a /0/009 MDCK Siat A/Hong Kong/988/009 a 9/0/009 MDCK Siat < TEST VIRUSES A/Norway/76/009 /0/009 MDCK Siat ND ND A/Singapore/0/009 b 6/0/009 MDCK Siat A/Moscow/9/009 /0/009 C Siat < ND ND A/Hong Kong/987/009 c 8/0/009 MDCKx Siat < ND ND A/Finland/5/009 a 0/0/009 M Siat < < ND ND A/Hong Kong/968/009 0/0/009 MDCKx Siat < 80 < ND ND A/Finland/57/009 0/0/009 M Siat < < ND ND A/St. Petersburg/66/009 06/0/009 E Siat < 80 0 < ND ND A/Menya/86/009 c /0/009 x Siat < < A/Norway/998/009 9/0/009 MDCK Siat < A/Singapore/7/009 0/05/009 MDCK Siat < A/Ghana/FS-09-78/009 a 08/05/009 x Siat < 0 < A/Singapore/5/009 08/05/009 MDCK Siat A/Aurillac/97/009 09/05/009 MDCK Siat < A/Ghana/FS-09-79/009 5/05/009 x Siat < < A/Ghana/FS-09-79/009 a /05/009 x Siat A/Singapore/5/009 /05/009 MDCK Siat < < < 60 < A/Ghana/FS-09-88/009 08/06/009 x Siat A/Iceland/8599/009 8/06/009 MDCK Siat < < A/Ghana/FS /009 0/06/009 x Siat A/Marseille/8/009 b 8/06/009 MDCK Siat < Algeria/G6/009 Jul-09 Cx Siat A/Hong Kong/78/009 0/07/009 MDCK Siat < < = <0; ND = not done Vaccine strain a. SN, G85A; b. N8S, G85S; c. H9N Page 5 of Haemagglutination inhibition titre Post infection ferret sera

16 Figure 6. Phylogenetic comparison of seasonal influenza HN HA genes Vaccine strains Reference strains Isolates Jan - Feb 009 Mar - Apr 009 May - Jun 009 NS A89T N8S G85S H9R H9N D5N K5R E7D hparis508d hhubeiyiling509japm hhongkong908n hnorway609ha hprague09j hparis7009ha hhongkong7608n hparma709j hkentucky0809cdcy hcaymanislands50809cdcu hbratislava909j hnewyork609cdca hguangdong509japm htoyama809japm hrabat0909had hincheon508had hmarrakech58909j hmarrakech5009haj hgyeonggibuk50008had hprague8909j hghanafs097909ha hghanafs097809ha hisrael009j hhk98809ha hcalifornia009cdca hhamamatuc98809japf hluxembourg08ha hmoscow09ha hfujiangulou6709cdca hhk97509ha htehran5909j hghazivin909haj hfinland509ha htehran009j hparaguay609cdcu hberlin5908had haragonrr0708ha hparaguay6009cdcu hparis7808d hflandersg7709f hdenmark08ded hengland55608n hparis0909ha hengland56508d hmarseilles809ha hkenya009cdcy hseychelles908u hchungbuk8508d hsingapore009ham hisrael09j hsingapore08d htexas808cdcn hcalifornia609cdca hjamaica96809cdca hwisconsin09cdcy htownsville09ausf hbrisbane09ausf hwashington009cdcj hnewyork5609cdcf hpennsylvania0808cdco hparis6508d hhk98709ha hmaryland0508cdcd hcameroon08508d hmenya8609ha hlisboa009j hstpetersburg08 VA SN G85A hnetherlands507oha hbrisbane5907eaus hhongkong87008cdcl hhongkong808o hhk85608l htianjinninnan5609japm hheilongjiang5809japf hgansuchengguan909cdcf hliaoning09cdcm hhongkong6506hal hthess05f newcal099ha hsolomon06haa Clade B Clade C Clade 0.0 Page 6 of

17 Figure 7. Phylogenetic comparison of seasonal influenza HN NA genes Vaccine strains Reference strains Isolates Jan - Feb 009 Mar - Apr 009 May - Jun 009 VM D8N EG RQ DN H6N E77G S66T N8S hthess05fna nncal099 GR H75Y H5N K78E G9K T87I K9E G5D S8P R0K M88I I67M L67I V9I T5I hhongkong908nn hparis508nd hprague09nj hparis7009na hnorway609naa hmarrakech5009nj hmarrakech58909nj hluxembourg08n hmoscow09na hparma709nj hbratislava909nj hhongkong7608nn hcaymanislands50809ncdcu hnewyork609ncdca hgyeonggibuk50008nd hhk98809na hfujiangulou6709ncdca hincheon508nd hcalifornia009ncdca hprague8909nf hhk97509na hghanafs097909nay hghanafs097809nau hrabat0909nj hisrael009nj htexas808ncdcn hwisconsin09ncdcy hcalifornia609ncdca hjamaica96809ncdca htownsville09nausf hbrisbane09nausf hpennsylvania0808ncdco hnewyork5609ncdcf hwashington009ncdcj hlisboa009nj hkentucky0809ncdcy hparaguay6009ncdcu hcameroon08508nd hmenya8609naa hparis6508nd hhk98709na hmaryland0508ncdcd hengland55608nn hengland56508nd hparis0909na hparis7808nd hflandersg7709nf hdenmark08nded hchungbuk8508nd hisrael09nj hseychelles908nu hsingapore08nd N7K Y00N htehran009nj hfinland509nam htehran5909nj haragonrr0708na hparaguay609ncdcu hmarseilles809nau hkenya009ncdcy hstpetersburg08n hberlin5908nd hliaoning09ncdcm hnetherlands507on hbrisbane5907njap Y00H K9E hhongkong6506nl nsolomon06ausg Clade B hhongkong87008ncdcl hhk85608nl hhongkong808no hgansuchengguan909ncdcf Clade Clade C 0.0 Page 7 of

18 Table 5. Antigenic analyses of influenza A HN viruses - Guinea Pig RBC Viruses Collection Passage A/Wis A/Trieste A/Bris A/Uru A/Fin/ A/Jhb A/Bris A/HK A/HK A/Perth A/Perth Date History 67/05 5c/07 0/07 76/07 9/08 5/08 /08 95/09 985/09 5/09 6/09 F/06 F5/07 F9/08 F6/08 F/08 F/08 F/09 F/09 F/09Aus FAus F REFERENCE VIRUSES A/Wisconsin/67/005 /08/005 SpfCkE E < 0 < < A/Trieste/5c/007 Jan-07 MDCKx Siat < A/Brisbane/0/007 06/0/007 E E A/Uruguay/76/007 /06/007 spfck, E E < < < < A/Finland/9/008 07/0/008 MDCK Siat A/Johannesburg/5/008 5/06/008 MDCKx Siat A/Brisbane//008 /06/008 E5 E < 0 < 0 A/Hong Kong/95/009 /0/009 MDCKx Siat A/Hong Kong/985/009 0/0/009 MDCKx Siat A/Perth/5/009 E E < < < < < < < A/Perth/6/009 E E < < < < < < < TEST VIRUSES A/Madagascar/007/009 6/05/009 MDCK Siat A/Madagascar/00/009 8/05/009 MDCK Siat A/Madagascar/86/009 0/06/009 MDCK Siat A/Ukraine/86/09 0/0/009 MDCK Siat ND ND A/Odessa/66/09 6/0/009 MDCK Siat ND ND A/Luxembourg/6/009 9/0/009 Siat ND ND A/Johannesburg//009 /05/009 MDCK Siat A/Johannesburg/6/009 6/05/009 MDCK Siat A/Johannesburg/9//009 0/06/009 MDCK Siat A/Lyon/CHU/0.56/009 /05/009 MDCK Siat < 0 < < A/Hong Kong/9/009 6/05/009 MDCKx Siat A/Hong Kong//009 7/05/009 MDCKx Siat A/Singapore/6/009 05/06/009 MDCK Siat A/Singapore/7/009 05/06/009 MDCK Siat A/Grenoble//009 7/06/009 MDCK Siat A/Luxembourg/78/009 08/05/009 MDCK Siat < A/Luxembourg/79/009 /05/009 Siat < ND ND A/Singapore//009 /05/009 MDCK Siat A/Johannesburg/6/009* /05/009 MDCK Siat < < = <0 Vaccine strains Haemagglutination inhibition titre Post infection ferret sera Page 8 of

19 Table 6. Antigenic analyses of influenza A HN viruses - Guinea Pig RBCs - 0nM Oseltamivir Haemagglutination inhibition titre Post infection ferret sera Viruses Collection Passage A/Wis A/Trieste A/Bris A/Uru A/Fin/ A/HK A/HK A/Perth A/Perth Date History 67/05 5c/07 0/07 76/07 9/08 95/09 985/09 5/09 6/09 F8/08 F5/07 F9/08 F6/08 F/088 F/09 F/09Aus FAus F REFERENCE VIRUSES A/Wisconsin/67/005 /08/005 SpfCkE \ < < < < A/Trieste/5c/007 Jan-07 MDCKx Siat A/Brisbane/0/007 06/0/007 E \ A/Uruguay/76/007 /06/007 spfck, E \ < < < < A/Finland/9/008 07/0/008 MDCK \ A/Hong Kong/95/009 /0/009 MDCKx \ 0 80 < A/Hong Kong/985/009 0/0/009 MDCKx \ < A/Perth/5/009 E \ < < < < < A/Perth/6/009 E \ < < < < < TEST VIRUSES A/Toulouse/955/009 9/0/009 MDCK Siat < A/Brisbane/5/009 07/05/009 E E < 0 ND ND A/Cape Town/9/009 /05/009 MDCK Siat < 0 < 0 A/Durban/675/009 Jun-09 MDCK Siat A/Madagascar/86/009 0/06/009 MDCK Siat A/Johannesburg/556/009 0/06/009 MDCK Siat < A/Lisboa//009 5/06/009 MDCK Siat A/England//009 0/07/009 Cx Siat < 0 ND ND A/Ghana/FS-05/009 /07/009 MDCK Siat ND ND A/Singapore//009 07/05/009 MDCK Siat 0 0 < A/Hong Kong/9/009 6/05/009 MDCK Siat A/Hong Kong//009 7/05/009 MDCK Siat 0 80 < A/Singapore//009 9/05/009 MDCK Siat 0 0 < A/Singapore/7/009 0/06/009 E E < < < < < ND ND A/England//009 0/06/009 Cx Siat < ND ND A/Scotland/5/009 0/06/009 Cx Siat ND ND A/Singapore/6/009 05/06/009 MDCK Siat A/Norway/79/009 0/06/009 MDCK Siat ND ND A/Grenoble//009 7/06/009 MDCK Siat 0 0 < A/Dakar/005/09 /06/009 MDCK Siat ND ND A/Hong Kong/9/009 0/07/009 MDCKx Siat ND ND A/Johannesburg/6/009* /05/009 MDCK Siat A/Victoria/08/009* 0/06/009 E E 80 < < 0 80 ND ND A/Johannesburg/85/009* 08/06/009 MDCK Siat < A/Mauritius/6/009* 0/07/009 MDCK Siat ND ND A/Mauritius/87/009* /07/009 MDCK Siat ND ND A/Ghana/FS-0/009* /07/009 MDCK Siat ND ND. < = <0; * characterised by K58N, N89K, TA Vaccine strains Page 9 of

20 Table 7. Antigenic analyses of influenza A HN viruses - Guinea Pig RBC - 0nM Oseltamivir Haemagglutination inhibition titre Post infection ferret sera Viruses Collection Passage A/Wis A/Trieste A/Bris A/Uru A/Fin/ A/Jhb A/HK A/HK A/Perth A/Wis A/Niig A/Niig Date History 67/05 5c/07 0/07 76/07 9/08 5/08 95/09 985/09 6/09 5/09 0/09 0/09 F/06 F5/07 F9/08 F6/08 F/088 F/08 F/09 F/09 F5/09 F/09 Jap F/09 Jap F5/09 REFERENCE VIRUSES A/Wisconsin/67/005 /08/005 SpfCkE E < < < < < < A/Trieste/5c/007 Jan-07 MDCKx Siat < 0 80 A/Brisbane/0/007 06/0/007 E E < < 0 < 0 0 A/Uruguay/76/007 /06/007 spfck, E E < < < < < < A/Finland/9/008 07/0/008 MDCK Siat < 0 0 < 0 0 A/Johannesburg/5/008 5/06/008 MDCKx Siat < 0 0 < 0 60 A/Hong Kong/95/009 /0/009 MDCKx Siat A/Hong Kong/985/009 0/0/009 MDCKx Siat A/Perth/6/009 0/07/009 E E < < < < < < A/Wisconsin/5/009 06/07/009 E E < < < 0 < < A/Niigata/0/009 MDCK Siat 80 < TEST VIRUSES A/Taiwan/0/009 E+ E < < < < < < A/Lithuania/K/009 9/0/009 Siat < 0 < < < 0 A/Iceland/60/009 0/0/009 MDCK Siat < 0 < < < < A/Lithuania/K/009 6/06/009 Siat < 0 80 < 0 60 A/Netherlands/76/009 6/06/009 xmdck Siat < 80 0 < 0 0 A/Ghana/FS-09-0/009 7/07/009 C Siat < < 0 A/Cameroon//009 0/07/009 Siat A/Cameroon/50/009 05/08/009 Siat A/Iran/755/009 Jun-09 MDCK Siat A/Columbia/7/009 0/05/009 C Siat < < A/Luxembourg/79/009 /05/009 MDCK Siat A/Mauritius/80/009 05/06/009 C Siat A/Mauritius/5/009 7/07/009 C Siat A/Lithuania/K/009 /07/009 Siat A/Ghana/FS-09-/009 0/08/009 C Siat < A/Ghana/FS-09-67/009 /08/009 C Siat < < < < = <0 Vaccine strains Page 0 of

21 Table 8. Antigenic analysis of influenza A HN viruses - Plaque Reduction Neutralization - MDCK-SIAT Viruses Collection Passage A/Well A/Bris A/Uru A/Eng A/Bris Date History /0 0/07 76/07 9/08 /08 F/0 F8/07 F/07 F/08 F/09 REFERENCE VIRUSES A/Wellington//00 6/0/00 E \ A/Brisbane/0/007 06/0/007 E \ A/Uruguay/76/007 /06/007 spfck, E \ A/England/9/008 /09/008 TC P \ A/Brisbane//08 /06/008 E TEST VIRUSES Virus Neutralization titre Post infection ferret sera A/Thessaloniki/6/009 5/0/009 p \ A/St. Petersburg/5/009 6/0/009 C \ A/Aragon/RR-8/009 9//008 MDCK \ A/Geneva/570509/009 /0/009 \ A/Geneva/588/009 0/0/009 \ A/Hong Kong/96/009 /0/009 MDCKx \ < A/Hong Kong/985/009 0/0/009 MDCKx \ < Based on 50% plaque reduction compared to serum negative controls Vaccine strain RDE treated Page of

22 Figure 8. Phylogenetic comparison of HN HA genes Vaccine strains Reference strains Isolates Feb-Mar 009 Apr-May 009 Jun-Jul 009 P0S A8S T8N K58R I60M R6Q VA E6K NK K58N N89K K58N N89K TA hscotland509ha hnorway7909hau hhawaii0709cdcm hdakar09y hengland09ha hmacedonia609ha hniigata67609japu hokinawa809japy hhk9509ha hhk509ha hhiroshima509japu hgrenoble09hau hsingapore909ausm hvictoria0909ausu hvictoria009ausu hnorway75909hau hsingapore709ha hsingapore09ha hluxembourg7809ha hhk98509ha hphilippines609ausm habudhabi09 hjamaica97009cdca hhk009ha hperth509ha hperth609ha hmauritius609hal hmauritius8709hal hghanafs009hal hvictoria0809ausu hshizuoka7609japy htaiwan709japy hjohanessburg8509hau hjohanessburg609hay hnorway09ha hnevada0609cdca hluxembourg6809ha hhawaii609cdca hestonia95609ha hlatvia909f hserbia5609ha hwalloniag8709f halbania5009ha hgeneva90909ha hflandersg79609f hathens809j hslovenia6009j hfinland509haa hvolos09j hkaliningrad09ha hwashington509cdca hserbia58009ha hmoldova6909m hjohanessburg55609hau hparis7509f hnorway6009ha hbarcelona09j hengland09ha VI hhonduras6909cdcy hghanafs0509hal hlisboa09hau hlyonchu05609hay hct9509ha hmadagascar8609ha hhongkong00009cdca hshanghai9009cdcm hbratislava09j hperugia09haj hlisboa009j hfinland5909haa hindiana009cdca hmadagascar6509ha hbrisbane08ausu ajohannesburg5008 hbrisbane007aus htrieste507j huruguay7607cdc hfinland908jha wisconsin67e05cdcg calif7e0cdcs HK/985 (Perth/6) clade Brisbane/0 clade 0.0 Page of

23 Figure 9. Locations of amino acid changes characteristic of A/Perth/6/009-like viruses on the H HA structure Page of

24 Figure 0. Phylogenetic comparison of HN NA genes Vaccine strains Reference strains Isolates Feb-Mar 009 Apr-May 009 Jun-Jul 009 P86H I6L S67N K69T I0V D6N ED I6T hhk9509na hhk09nau hhawaii0709ncdcm hnorway75909nu hbcolumbiarv09ncdcm hscotland509n hvictoria009nausu hmacedonia609nau hsingapore909nausm hphilippines609nausm hvictoria0909nausu hjamaica97009ncdca hnorway7909nu hluxembourg7809nay hsingapore709nau hgrenoble09nu hhk98509na hsingapore09nay hengland09n hhk009nau hperth609na hfinland5909na hnorway09nj hlisboa009nj hserbia5609naj hgeneva90909na hwalloniag8709nf hbratislava709nj hwashington509ncdca hgeneva na hflandersg79609nf hslovenia6009nj hmoldova6909na hfinland509na hvolos09nj hparis7509na hathens809nj halbania5009na hserbia58009naf hkaliningrad09na hbelgiumg0809nj hlyonchu05609ny hct9509n hghanafs0509nl hlisboa09nu I77K hluxembourg6809naf hnevada0609ncdca hnorway09nm hlatvia909nf hhawaii609ncdca hestonia95609naf hbrisbane08nausu hlatvia0709nf hindiana009ncdca Y0C hvictoria0809nausu hjohanessburg609ny hghanafs009nl hmauritius8709nl hmauritius609nl hjohanessburg8509nu hparis7509na hjohanessburg55609nu hengland09n hbarcelona09nj hlatvia09nj hnorway6009naf hhongkong00009ncdca hshanghai9009ncdcm hbratislava09nj hperugia09nj hmadagascar6509naa hmadagascar8609nau hhonduras6909ncdcy hjohannesburg508n hfinland907n hbrisbane007naus htrieste507cn huruguay7607ncdc hwisconsin6705gna ncalifornia70cdcs HK/985 (Perth/6) clade Brisbane/0 clade 0.0 Page of

25 Table 9. Antigenic analyses of influenza B viruses (Victoria lineage) Haemagglutination inhibition titre Post infection ferret sera Viruses Collection Passage B/Shan B/Shan B/Bris B/HK B/Mal B/Vic B/Eng B/Bris B/Bris B/Paris date History 7/97 7/97 /0 5/05 506/0 0/06 9/08 /08 60/08 76/08 F/0 F/0 F5/05 F8/05 F6/06 F/08 F/09 F/09 F/09 REFERENCE VIRUSES B/Shandong/7/97 Ex < < B/Brisbane//00 0/07/00 E \ < 0 < B/HK/5/005 07/0/005 MDCK Siat < < < B/Malaysia/506/00 06//00 E \ < < B/Victoria/0/006 06/06/006 E \ B/England/9/008 9/08/008 E \ < < B/Brisbane//008 /07/008 E \ < B/Brisbane/60/008 0/08/008 E \ B/Paris/76/008 09/0/009 C Siat 0 < < < < TEST VIRUSES B/Iwate/7/009 MDCK+ Siat 60 < 0 60 < 0 < < < < B/Hubei-xiling/7/009 E+ E < B/Sabac/060/009 0/0/009 Primo Siat < < ND B/Belgrade/09/009 0/0/009 MDCK Siat < 0 ND B/Hong Kong/8/009 06/0/009 MDCKx Siat < 0 ND B/Singapore//009 0/05/009 MDCK Siat 60 < < < B/Hong Kong/5/009 09/07/009 MDCKx Siat 0 < 0 80 < < < < < < B/Okinawa/0/009 MDCK+ Siat 0 < < 0 < < B/Sabac//009 /0/009 MDCK Siat 60 0 < < 0 80 B/St. Petersburg/0/009 5/0/009 C Siat < < 0 0 < 0 80 B/Georgia/07 (9G)/009 0/0/009 CII Siat 0 < 0 < B/Georgia/0 (G)/009 6/0/009 CII Siat 0 < 0 < ND B/Ukraine/0/09 05/0/009 MDCK Siat 0 < 0 < < 0 ND B/Geneva/77/009 /0/009 x Siat 0 < < < 0 < < < 80 ND B/St. Petersburg/6/009 6/0/009 E E < ND B/Zagreb/596/009 0/0/009 E E < ND B/Zagreb/6009 0/0/009 E E < ND B/Iceland/7/009 0/0/009 MDCK Siat 80 < B/Netherlands/75/009 07/0/009 Mk Siat 0 < < 0 < < ND B/Kiev/95/09 5/0/009 MDCK Siat ND B/Finland/5/009 6/0/009 MDCK Siat < ND B/Madagascar/77/009 9/0/009 MDCK Siat 0 < B/St. Petersburg/5/009 0/0/009 E Siat < ND B/Norway/85/009 0/05/009 MDCK Siat 0 < < 0 < < B/Norway/87/009 08/05/009 MDCK Siat 0 < < < < < B/Norway/7/009 8/05/009 MDCK Siat 0 < < < 0 < 0 < 60 ND B/Madagascar/5/009 0/06/009 MDCK Siat 60 < < 0 0 < < < 80 ND B/Iceland/86/009 9/06/009 MDCK Siat 0 < < < < < B/Ghana/FS-08/009 /07/009 MDCK Siat 60 < B/Mauritius/55/009 8/07/009 MDCK Siat 0 < < < < < < = <0;. hyperimmune sheep serum Page 5 of Vaccine strain

26 Figure. Phylogenetic comparison of influenza B HA genes (Victoria lineage) Vaccine strain Reference strains Isolates Jan - Feb 009 Mar - Apr 009 May - Jul 009 K8E K80R K9N S7P bris0ausl tehran800f beijing96 shandong797 N75K N65K A0V L58P V6I V5I blatvia78809f btexas509cdcf btexas09cdcf bbelgiumg08609han bparis7609f bavila09f btexas0909cdcm bindiana09cdcf bserbia09ham bnorway0809ha bmontana009cdca bbayern608o bnorway0609ha bbrisbane08ausl bengland908eg bgeorgai509f bgeorgia60-g09a bshizuoka09japj bnorway709ha bmoldova6809m bflandersg8809f bmagnisie709j bathens809j bparis709frf bnorway85009u bmauritius5509l bbrisbane6008ausg bminnesota009cdca bsapporo909japa boregon09cdcf btennessee009cdca bhawaii009cdcm bokinawa009japy bfinland509a bmadagascar5509ha bmadagascar509ha bmadagascar55009hau bbratislava0809j bbadenw5808o bbeijingxicheng509cdcm bghanafs0709l bbarcelona9808n bjohannesburg08u bagadir508had btanger509d bvalladolid08haj bparis709f bparma508haf bsingapore08n bshiraz09haf bvictoria006hau bguangxingning09japa bhunanlingling809cdca balgeriag08 bhongkong708d bhubeisongzi508cdcn bjiangxiguuixi6609cdcm bhubeixiling509cdcm btrieste808haj bserbia57909haf bserbia09m bsabac9009haj bsabac8909haj bhongkong99808m bjiangsugulou509cdcf btochigi508japo byamagata509japa bminnesota0609cdcy bwisconsin009cdcf bwisconsin09cdcf N65K bisrael09j bisrael09j malaysia5060ausd bhk505f bnorthdakota09cdcf bsingapore09y Brisbane/60 Clade 0.0 Page 6 of

27 Figure. Phylogenetic comparison of influenza B NA genes Vaccine strain Reference strains Isolates Jan - Feb 009 Mar - Apr 009 May - Jul 009 PQ T9I T68A N98S N9K N5D SP E7K A89T D9E F96L E6T D0E N0K N9D E0K Q6H L7F SP N5K N9D D6N N5K G8E K86R N5T N0D bserbia09nam bnmontana009cdca bnorway0609naa bnengland908eg bnbrisbane08ausl bnhawaii009cdcm bnorway85009nau bbrisbane6008ausg bnminnesota009cdca bgeorgia60-g09naa bmauritius5509nal bnoregon09cdcf bflandersg8809naf bmoldova6809nam bnorway709nay bathens809naj bparis7609naf bghanafs0709nal bbelgiumg08609nan bntexas09cdcf bntexas0909cdcm bfinland509naa blatvia78809naf bavila09naf bnindiana09cdcf bbratislava0809naj bmadagascar5509nau bnbeijingxicheng509cdcm bbarcelona9808nan bvalladolid08naj btanger509nad bnvictoria006ausu G78E D9N A58E K7Q E0K DG A65T QR D6N bnsingapore08n bnnorthdakota09cdcf bnwisconsin09cdcf bntennessee009cdca bnwisconsin009cdcf bnminnesota0609cdcy bisrael09naj bhongkong99808nam bnjiangsugulou509cdcf bserbia57909naf bnhubeisongzi508cdcn bnjiangxiguuixi6609cdcm bnhubeixiling509cdcm bnhongkong708s bnhunanlingling809cdca bnmalaysia5060 bhk505f bsingapore09nay bnghanafs8508n bnhongkong09cdca bvalladolid808naf bntexas708cdcd bnmissouri009cdcf bnminnesota009cdca bnghanafs808d bghanamh08nas bengland508na M75K PS T8M A89T S97R bnbayern708n bnnetherlands7508s bnhongkong708s bnmexico7609cdcm bnvirginia009cdcm bnnewmexico009cdcj bswitzerland908nad begypt09naj bntexas09cdcm bnwashington008cdcd bnhongkong609cdcm bncalifornia009cdcj bcameroon08908nad bnbangladesh07cdcg egypt05j bnflorida70 bnflorida06 bmendozar5008na bnbrisbane07ausg bnhawaii009cdcm bnshand797 Yamagata-lineage HA Victoria-lineage HA 0.0 Page 7 of

28 Table 0. Antigenic analyses of influenza B viruses (Yamagata lineage) Viruses Collection Passage B/Fl B/Eg B/Fl B/Bris B/Eng B/Vall B/Ban date History /06 /05 /06 /07 5/08 8/08 /07 Sh 78 F/07 F/07 F/07 F9/08 F0/08 F/08 REFERENCE VIRUSES B/Egypt//005 0/05/005 E E B/Florida//006 5//006 E E B/Brisbane//007 0/09/007 E E B/England/5/008 Ex E 60 < < 0 0 < 0 B/Valladolid/8/008 8/0/008 MDCK Siat B/Bangladesh//007 9/08/007 E E TEST VIRUSES B/Latvia/-0/009 0/0/009 MDCK Siat B/Mashad/6/009 08/0/009 Siat B/Zahedan/7/009 /0/009 Siat B/Aswan/895/009 5/0/009 Cx Siat B/Hong Kong/05/009 0/0/009 MDCKx Siat B/Alexandria//009 0/0/009 Cx Siat B/Alexandria//009 /0/009 Cx Siat B/Aswan/978/009 07/0/009 Cx Siat B/Alexandria/9/009 0/0/009 Cx Siat B/Aswan/98/009 /0/009 Cx Siat B/Tehran/8/009 6/05/009 Siat B/Norway/0/009 0/05/009 MDCK Siat B/Stockholm//009 0/05/009 Siat B/Hong Kong/5/009 /07/009 MDCKx Siat < = <0;. hyperimmune sheep serum Haemagglutination inhibition titre Post infection ferret sera Page 8 of

29 Figure. Phylogenetic comparison of influenza B HA genes (Yamagata lineage) Reference strains Isolates Dec Jan 009 Feb - Mar 009 Apr - May 009 N0S S50I N65Y G9D R8K P08A bminnesota009cdca bmissouri009cdcf btexas708cdcd bmexico7609cdcm bnewmexico009cdcj btexas09cdcm bghanamh08s bnorway7808hau bsalta7008haj bncarolina009cdcf bmichigan08cdcn bhk0808hau blyon0608u bhk608hal bhongkong708s balgeriag7608 bmashad609yha begypt09haj bnetherlands7508s bengland508ha balaska0508cdcn bnorway508f bakita08japn brzeszow08m btehran809yha bhongkong609cdcm bzahedan709yhaf bvalladolid808haf blatvia608a bmures6008f bghanafs8508n bhongkong09cdca bswitzerland908had bcalifornia009cdcj bdakar608hag bdakar08hal bghanafs5008s bsingaporen608hay bmauritius8708l bghanafs708had bparis68009j bparis68509f biceland808m bbayern708n bjohannesb08hal bwashington008cdcd blatvia009f bvirginia009cdcm bireland08m bcameroon08908d bbangladesh07gcdc bnorway0908hay balaska008cdcn bhongkong908a bsingaporeen07808han bulanude508a bsingaporen08hay bbrisbane007ausg egypt05j bkawasaki08japn G9S bflorida70hao K7R Brisbane//07 clade bniigata608japd bhawaii009cdcm bbsas896008u bbolivia8808cdcn btaiwan9908japn bmendozar5008ha bflorida06cdcn bbratislava08haa blaplata55808u bpavia08its Bangladesh//07 clade Florida//06 clade 0.0 Page 9 of

30 Figure. Phylogenetic comparison of influenza H5 HA genes Candidate Vaccine strains 009 viruses P7S D97N H0R WP R0G SP FY R6K N65H A8E M6V Q9H N09S EK K7R A7I Y5N RG K7R N55D N9D T5I N5D DN S0N 9 I5T P5S S57F h5ckegyptnic909ha h5ckegyptnic509ha aegyptn08009 aegyptn aegyptn aegyptn h5egypt56namru08db aegyptn aegyptn08009 h5egypt07db h5egypt907806ha h5ckegypt7098vir0807dbha h5ckisrael05508dbha h5egypt00namru08db h5ckegyptc07ha h5tyturkey05ha h5nigeria6e07ha h5cknigeria07507dbha h5cknigeria807ha h5ckcotedivoire78706db h5cksudan78706hadb h5turkey506e h5iraq06eha h5hsmongolia05hadb h5tyengland5007db T59I V9I h5chickentogop08ha h5chickentogop508ha h5cktogo0607dbha h5ckniger07506db h5ostrichnigeria07506db h5swanscotland006db h5dkgermanyr0807dbha h5ducknigeria708db h5ckkrasnodar0007db achickenbang h5pakistan8t07ha h5ckindianiv8706dbha h5azer0606ha h5bhgooseqinghaia05db h5indonesia8808dbha h5indonesia7708dbha h5indonesiacdc007db h5indonesia505hadb h5dkkprogobbvet90cdc h5indonesia605ha R6I A96S G7S R5K D90N h5dkhunan7950db h5anhui05db h5dklaos9506hadb h5sichuan06db h5cmagpiehk6506db h5pengland905ha h5ckvietnamncvd507dbha h5falconhongkong08dbha h5cklaospo707dbha h5dkvietnamncvd5907dbha h5dkvietnamncvd07dbha h5jpwhiteeyehk0806db h5ckmalaysia507dbha h5dkhunan50db agulltyva509 agrebetyva009 h5buzzardhk907dbha h5ckprimorsky8508dbha h5wswanakita08dbha aguangxi009 h5robinhk09708dbha h5maghongkong50507db h5dkvietnamncvd907dbha h5cambodiar db h5ckvietnamncvd07dbha h5vietnamjp6-05jap h5ckthailandns908dbha h5ckthailandicrc6808dbha h5vietnam90cdc h5vietnam00cdc h5hk0 h5gsguiyang706db h5envqinghai08dbha Clade Clade. Clade.. Clade. Clade.. h5ckvietnamncvd0608dbha h5ckvietnamncvd008dbha Clade Clade Page 0 of

31 Figure 5. Phylogenetic comparison of influenza H5 NA genes Candidate Vaccine strains 009 viruses Q9L GR P0L KE V6I D70N ED I8M V5A HR N7K S9V P0S V89G RC N8S KR QK VI h5dkhunan7950dbn S9F A6D h5ckegypt5nic09na h5ckegypt9nic09na aegyptn08009 aegyptn aegyptn aegyptn h5egypt56namru08dbn h5egypt07dbn aegyptn aegyptn08009 h5egypt907806n h5ckegyptc07n T88I h5ckegypt7098vir0807dbn h5ckisrael05508dbn h5egypt00namru08dbn h5tyturkey05n h5turkey506n h5iraq06n h5tyengland5007ndb h5ckci78706ndb h5cksudan78706ndb h5hsmongolia05ndb G8E V89M LM S50G RK h5chickentogop08na h5chickentogop508na h5cktogo0607dbn h5cknigeria07506ndb h5cknigeria807n h5cknigeria07507dbn h5ostnigeria07506ndb h5nigeria6e07n h5bhgooseqinghaia05ndb h5swanscotland006ndb h5ducknigeria708dbn h5dkgermanyr0807dbn h5ckkrasnodar0007ndb h5pakistan8t07n h5ckindianiv8706dbn h5azer0606n h5ckvietnamncvd0608dbn h5cambodiar ndb h5ckvietnamncvd07dbn h5dkvietnamncvd907dbn h5dkvietnamncvd07dbn h5vietnamjp6-05jap h5ckthailandns908dbn h5ckthailandicrc6808dbn h5viet90ndb h5viet00ndb h5envqinghai08dbn h5hk0db h5gsguiyang706dbn h5ckprimorsky8508dbn h5wswanhokkaido08dbn agrebetyva009 agulltyva509 h5buzzardhk907dbn h5maghongkong50507dbn h5robinhk09708dbn h5ckvietnamncvd507dbn h5cklaospo707dbn h5anhui05ndb h5dklaos9506ndb h5pengland905n h5cmagpiehk6506dbn h5sichuan06ndb h5ckmalaysia507dbn h5dkhunan50db h5dkvietnamncvd5907dbn h5falconhongkong08dbn h5jpwhiteeyehk0806dbn h5indonesia8808dbn h5indonesia7708dbn h5indonesiacdc007ndb h5indonesia505db h5dkkprogobbvet90cdc h5indonesia605hkn Clade. Clade Clade. Clade 7 Clade Clade.. Clade Page of