WHO INFLUENZA CENTRE LONDON

Transcription

1 WHO INFLUENZA CENTRE LONDON Report February 2009

2 WHO Collaborating Centre for Reference and Research on Influenza National Institute for Medical Research The Ridgeway Mill Hill London, NW7 1AA 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: 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 3 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.

3 Influenza activity, October 2008 to March 2009 Influenza was in general mild. Influenza activity in most parts of the world was lower compared with the same period last year, while in Europe it was higher than during the previous two seasons (Figure 1). In the southern hemisphere, influenza activity continued into November; influenza B viruses had predominated in Australia and New Zealand during September, while influenza A(H1N1), A(H3N2) and B viruses circulated in different countries to varying extents. In Europe, influenza activity commenced in October (Figure 1) and followed a west to east progression. A(H3N2) viruses predominated for most of the season, with influenza B viruses increasing during February and March. H3N2 viruses also predominated in Japan, whereas A(H1N1) viruses were relatively more prevalent in other Asian countries, such as Korea. In north African countries A(H3N2) and A(H1N1) viruses co-circulated, while in some west African countries A(H3N2) or B viruses predominated. In North America A(H1N1)viruses predominated in the USA, while B viruses predominated in Canada. Antigenic and genetic characteristics of influenza A(H1N1), A(H3N2) and B viruses isolated during October 2008 to February Of approximately 550 human influenza viruses, isolated in 33 countries during October 2008 to February 2009, the majority(68%) were A(H3N2) and 20% were A(H1N1) (Table 1). Influenza B viruses accounted for 12% of the viruses received, including similar numbers of B/Victoria lineage and B/Yamagata lineage viruses; viruses from African countries were of the B/Yamagata/16/88 lineage, whereas most of those from European countries were of the B/Victoria/2/87 lineage. A(H1N1) viruses In HI tests, the majority of H1N1 viruses were antigenically closely related to the MDCK cellgrown reference viruses A/Netherlands/345/2007 (clades 2B), A/Seychelles/2239/2008 (clade 2B), A/Hong Kong/1856/2008 (clade 2C) and/or A/Hong Kong/1870/2008 (clade 2C) (Table 2). Antisera to these viruses did not distinguish between clade 2B and 2C HAs. HI titres with antisera to the egg-grown vaccine virus A/Brisbane/59/07 were in general reduced relative to the homologous titre, apparently reflecting differences between the HAs (e.g. an amino acid substitution at position 186, D186N/V) of cell culture and egg isolates. No A/New Caledonia/20/99-like viruses were identified. The majority of HA and NA sequences fell within clade 2B, represented by A/Brisbane/59/2007 (Figures 2 and 3). Most clade 2B HA sequences of recent isolates possess the common amino acid change A189T, but fall into subgroups distinguished by different signature amino acid changes S141N+G185A, S141R+N183D, N183S+G185S/N (+N244S), G185V+H192R or H192N (Figure 2, Table 3). NA sequences fell into corresponding subgroups within clade 2B (based on nucleotide sequences, but were not distinguished by amino acid differences) (Figure 3). The majority of NAs possessed the H275Y mutation, conferring oseltamivir resistance, and the D354G substitution (Figure 3).

4 Relatively few HA and NA sequences (mainly of viruses from China) fell within clade 2C, represented by A/Hong Kong/1856/ Antiviral susceptibility The majority of H1N1 viruses tested were shown to be resistant to oseltamivir, either by enzyme inhibition assay or the presence of the H275Y mutation in NA, or both (Table1). Data produced by the EISS/VIRGIL network showed that 97% of European isolates tested were resistant to oseltamivir, but retained sensitivity to zanamivir and amantadine/rimantadine. A(H3N2) viruses There was a marked increase in the proportion of H3N2 viruses which failed to agglutinate turkey or human red blood cells (RBCs), but agglutinated guinea pig RBCs, such that the majority of recent isolates received from some countries agglutinated only guinea pig RBCs. As previously, various HI tests using turkey RBCs (Table 4) continued to show that most H3N2 viruses gave patterns of reduced HI titres similar to those observed for MDCK cell-grown reference viruses, such as A/Trieste/25c/2007 and/or A/Johannesburg/15/2008, which also gave relatively low homologous HI titres with their corresponding ferret antisera, compared with eggpassaged reference viruses. Furthermore, antisera raised in ferrets infected with MDCK cellgrown or egg-grown reference viruses gave similar patterns of differential HI reactivity with the various reference viruses, indicating that the cell culture-grown reference viruses and egg-grown vaccine viruses elicited similar antibody responses in ferrets. HI tests of viruses which failed to agglutinate turkey (or human) RBCs, with guinea pig RBCs (Table 5) gave high HI titres, comparable to those obtained with egg-grown reference viruses and showed that most of the viruses were antigenically closely related to the vaccine strains A/Brisbane/10/2007 and A/Uruguay/716/2007. Neutralisation tests did not distinguish between egg-grown (e.g. A/Brisbane/10/2007) and MDCK cell-grown (e.g. A/Johannesburg/15/2008) reference viruses. They showed that for several of the viruses which gave relatively low HI titres (blue in Table 4), as for some which agglutinated only guinea pig RBCs (blue in Tables 5 and 6), neutralization titres of reference antisera were similar to those with the homlogous reference viruses, indicating that the viruses were antigenically closely related to the A/Brisbane/10/2007 and A/Uruguay/716/2007 vaccine strains (Table 6). The majority of HA sequences fell within the phylogenetic group characterised by the change K173Q relative to the sequence of A/Brisbane/10/2007 (Figure 4). Sequences were fairly homogeneous and no prominent phylogenetic subgroups were distinguished by additional common amino acid substitutions. The majority of NA sequences fell within a corresponding group defined by the substitutions D147N and I215V, relative to the sequence of A/Brisbane/10/2007 (Figure 5). The sequences of many recent European isolates fell within a subgroup characterized by the additional changes P386H and I464L (the corresponding HA sequences did not possess any common amino acid differences).

5 Antiviral susceptibility All viruses analysed carried asparagine at position 31 in M2, indicative of resistance to amantadine and rimantadine, and were sensitive to oseltamivir and zanamivir. B viruses In HI tests, the majority of B/Victoria-lineage viruses were shown to be antigenically closely related either to the MDCK cell-grown reference virus B/Hong Kong/45/2005 (B/Malaysia/2506/2004-like) or to B/Brisbane/60/2008 and B/Brisbane/33/2008, the strains recommended by WHO for inclusion in the vaccine (Table 7). Reduced HI titres, relative to those with the homologous egg-grown reference viruses reflect differences commonly observed between the reactivities of egg- and MDCK cell-grown viruses. The majority of HA sequences of recent isolates fell within clade 2, characterized by the changes N75K, V146I, N165K and N172P relative to the HA of B/Malaysia/2506/2004 and represented by B/Brisbane/60/2008 and B/Brisbane/33/2008 (Figure 6, Table 9). NA sequences of these viruses fell within clade 2A and were characterized by additional changes I204V, D329N and A358E, relative to the sequence of B/Victoria/304/2006 (Figure 7, Table 9). NA sequences of some recent isolates with clade 1 HAs, such as B/Brazil/2937/2008, fell within clade 2B (Figure 7). HI tests of the B/Yamagata-lineage viruses showed that many were antigenically closely related to the vaccine strains, B/Florida/4/2006 and B/Brisbane/3/2007, whereas others were more closely related to the recent reference viruses, B/Barcelona/143/2008 (MDCK), B/England/145/2008 (egg), B/Valladolid/18/2008 (MDCK) and/or B/Bangladesh/3333/2007 (egg), (Table 8). HA sequences of recent B/Yamagata-lineage isolates were similar to those of viruses isolated during 2007 and 2008 and fell into one of the 3 principal clades, with most falling within clade 3, represented by e.g. B/Bangladesh/3333/2007 and B/England/145/2008 (Figure 8, Table 9). HAs in the three clades were not distinguished in HI tests. NA sequences fell into corresponding phylogenetic subgroups, as previously; those represented by the vaccine viruses (clades 1 and 2) were similar and distinguished from those of clade 3 by 7 amino acid differences, including the substitutions D463N and A465T, relative to the sequence of B/Bangladesh/3333/2007 (Figure 7, Table 9).

6 Figure 1: Influenza situation in Europe season (weeks 40/08-09/09)

7 Table 1. Human influenza isolates, October February 2009 Country H1N1 H3N2 B Yamagata Victoria lineage lineage October Argentina 1 1 Cameroon France 5 Germany Ghana Hong Kong Madagascar 2 2 Norway 1 Spain 2 1 Sweden 4 UK November Algeria 1 Cameroon Belgium 1 1 Denmark 2 Egypt 6 France Germany 2 1 Ghana 7 Hong Kong Italy 6 1 Madagascar 2 Mauritius 1 Norway Portugal 1 Romania 1 Singapore Spain 4 Sweden Switzerland 1 UK December Algeria 8 Belgium 22 1 Cameroon Finland 6 France Germany Ghana 3 Israel Italy 16 Korea Latvia 4 Luxembourg 1? 4 Morocco Norway Portugal 4? 12 Singapore 1 1 Slovakia 1 Spain 34 Sweden Switzerland 10 1 UK January 2008 Belgium Czech Republic 3? 12 Egypt 1 Germany 2 Greece 8 3 Iran 2 2 Israel 4? 8 3 Italy 5? 14 Latvia 2 Morocco Norway 2 Portugal 2? 1 Romania 9 Slovakia 1? 6 1 Slovenia 3 Spain 11 Switzerland Tunisia 2 February 2009 Czech Republic 1? 2 Total = % 68% 69 (12%) oseltamivir resistant;? = yet to be determined

8 Table 2. Antigenic analyses of influenza A H1N1 viruses Viruses Collection Passage A/NC A/HK A/SI A/Neth A/Bris A/SP A/HK A/Sey A/HK Date History 20/ /06 3/06 345/07 59/07 12/ / / /08 F19/08 F14/06 F8/07 F23/07 F6/08 F17/08 F28/08 F27/08 F4/09 REFERENCE VIRUSES Haemagglutination inhibition titre 1 Post infection ferret sera A/New Caledonia/20/99 06/09/1999 Ex 640 < < A/Hong Kong/2652/ /07/2006 E2 \ A/Solomon Islands/3/ /08/2006 E3 \ A/Netherlands/345/ /10/2007 MDCK2 \ A/Brisbane/59/ /07/2007 E2 \ A/St.Petersburg/12/ /02/2008 E2 \ A/Hong Kong/1856/ /07/2008 MDCK2 \ < A/Seychelles/2239/ /06/2009 MDCK1 \ A/Hong Kong/1870/ /07/2008 MDCK4 \2 < TEST VIRUSES A/England/556/ /11/2008 MDCK P1 \ < ND A/Singapore/43/ /12/2008 MDCK1 \1 < A/Lisboa/29/ /12/2008 MDCK \ A/Incheon/4532/ /12/2008 MDCK-P2 \1 < A/Luxembourg/1344/2008 unknown MDCKx \ ND A/Gyeonggibuk/4500/ /12/2008 MDCK-P2 \1 < A/Incheon/4532/ /12/2008 MDCK-P2 \1 < A/Hong Kong/3176/ /11/2008 MDCK2 \1 < A/Paris/781/ /12/2008 C1 \ A/Rabat/109/09 01/12/2008 Cx \3 < < < A/Marrakech/510/ /01/2009 Cx \2 < A/Berlin/59/ /12/2008 MDCK4 \1 < A/Sachsen-Anhalt/54/ /12/2008 MDCK4 \1 < A/Parma/25/09 12/01/2009 MDCK1 \1 < A/Parma/27/09 14/01/2009 MDCK1 \1 < 40 < A/Fes/213/09 14/01/2009 Cx \ A/Israel/63/08 15/12/2008 MDCKx \1 < A/Israel/64/08 19/12/2008 MDCKx \ A/Norway/2262/ /12/2008 MDCK1 \ A/Sweden/8/08 09/12/2008 MDCK1 \ ND A/Pilsen/44/ /01/2009 MDCK3 \1 < A/Prague/89/ /02/2009 MDCK2 \1 < A/Cameroon/08-224/ /12/2008 MDCKx \1 < A/Cameroon/08-232/ /12/2008 MDCKx \ A/Laayoune/208/09 17/01/2009 Cx \1 < < = <40; ND = not done Vaccine strain

9 Figure 2. Phylogenetic comparison of influenza H1N1 HA genes Vaccine strain Reference strains Isolates October 2008 November 2008 Dec Jan 2009 K140E R208K T266N R188K D35N K145R E273K G185V H192R N244S S141N G185A A189T N183S G185S/N S141R N183D H192N R188M S36N T193K A189T h1hongkong317608n h1norway208908nwn h1norway216108han h1sakai3008japn h1hongkong319208n h1paris44508d h1incheon453208had h1rabat10909had h1ghanafs30908o h1luxembourg134408ha h1hiroshima4808japn h1sapporo8308japd h1ghana16808l h1fes36309had h1dakar1408g h1taiwan63908cdcd h1mie3708japd h1maryland0708cdcd h1gyeonggibuk450008had h1paris22408frn h1marrakech51009haj h1yokohama9608japd h1idaho2308cdcd h1texas1808cdcn h1washington0109cdcj h1newjersy2308cdcd h1pennsylvania0908cdcn england41108hao h1denmark21408ded h1paris78108d h1england56508d h1johannesburg34008l h1minnesota2708cdcd h1berlin5908had h1england55608n h1seychelles223408u h1mauritius40308l h1singapore10408cdcd A/Seychelles/2239/2008 h1paris24408frn h1hongkong321208cdcd h1nebraska0708cdcd h1singapore4308d h1buenosaires g buenosaires130608hay h1florida2808cdcd salta209308has h1ghanafs21608g h1jilin119108japn h1paris64508d h1maryland0508cdcd h1lisbon2908pgd h1cameroon07308y A/St. Petersburg/12/2007 h1brazil105908cdcu h1chubut u h1buenosaires1308y h1singapore4008n h1capetown12408l A/Netherlands/345/2007 h1egypt1007oha A/Brisbane/59/2007 h1madagas236008u h1ghana18908l neuquen has h1hk140408l I47K E68G K82R h1tianjin112008cdco h1hongkong314808o h1taiwan64808ausn A/Hong Kong/1856/2008 h1anhui151408cdco h1california0708cdcs h1shanxi113408cdcn h1beijing19008cdcn A/Hong Kong/2652/2006 h1stpeter1007jha h1fukushima14106haj A/Solomon Islands/3/2006 A/Thessaloniki/24/2005 A/New Caledonia/20/99 Clade 1 Clade 2A Clade 2B Clade 2C 10 nucleotide substitutions

10 Figure 3. Phylogenetic comparison of H1N1 neuraminidase genes Vaccine strain Reference strains Isolates October 2008 November 2008 Dec Jan 2009 * Oseltamivir resistant V234M D382N E214G R222Q D344N H64N E77G S266T H45N K78E G249K T287I K329E G354D S82P R130K M188I I267M L367I V393I T453I V149A A/Thessaloniki/24/2005 A/New Caledonia/20/99 h1norway208908n1nwn h1norway216108n1nwn h1hongkong317608n1n h1paris22408n1frn h1maryland0708n1cdcd h1hiroshima4808n1japn h1mie3708n1japd h1taiwan63908n1cdcd h1gyeonggibuk450008n1d h1mie3208n1japn h1sakai3008n1japn h1marrakech51009n1j h1luxembourg134408n1 h1dakar1408n1g h1hongkong319208n1n h1paris44508n1d h1ghana16808n1l h1incheon453208n1d h1ghanafs30908na h1ghanafs20508n1l h1cameroon07308n1y h1rabat10909n1j h1idaho2308n1cdcd h1texas1808n1cdcn h1pennsylvania0908n1cdcn h1newjersy2308n1cdcd h1washington0109n1cdcj h1buenosaires nag h1florida2808n1cdcd h1salta209308nas h1buenosaires130608naj h1algeriag86408n1m h1paris64508n1d h1ghanafs21608n1g h1maryland0508n1cdcd h1england55608n1n h1england56508n1d h1paris78108n1d h1england41108nao h1denmark21408n1ded A/Seychelles/2239/2008 h1singapore4308n1d h1mauritius40308n1l h1seychelles223408n1u h1hongkong321208n1cdcd h1paris24408n1frn h1sweden808n1sed h1berlin5908n1d h1johannesburg34008n1l h1sweden1008n1sed h1taiwan62508n1japn h1capetown12408n1l h1chubut n1u h1nebraska0708n1cdcd h1singapore10408n1cdcd A/St. Petersburg/12/2008 h1singapore4008n1n h1bsasr1308n1m h1brazil105908n1cdcu A/Netherlands/345/2007 h1egypt1007n1 D354G H275Y A/Brisbane/59/2007 h1ghana18908n1l h1neuquen nas h1madagascar236008n1u h1beijing19008n1cdcn h1shanxi113408n1cdcn Y100H K329E T264I A/Hong Kong/2652/2006 h1stpeter1007jn1 h1fukushima14106n1j A/Solomon Islands/3/2006 h1anhui151408n1cdco h1hk140408n1l h1california0708n1cdcs h1tianjin112008n1cdco A/Hong Kong/1856/2008 h1hongkong314808n1o h1taiwan64808n1ausn Clade 1 Clade 2A * Clade 2B Clade 2C 10 nucleotide substitutions

11 Table 3. Amino acid changes characteristic of the recent principal H1N1 sequence variants Clade Representative strain Amino acid changes 1 HA NA 2 2B A/Seychelles/2239/2008 D35N H45N (A/Brisbane/59/2007) K145R K78E R188K G249K E273K T287I A189T K329E (S141N + G185A)* H275Y (N183S + G185S/N)* (V149A) (S141R + N183D)* (G185V + H192R)* (H192N)* (N244S)* 2C A/Hong Kong/1856/2008 S36N S82P I47K Y100H E68G R130K K82R M188I R188M I267M A189T K329E T193K L367I V393I T453I (T264I) 1. Relative to A/Solomon Islands/3/2006 (Clade 2A) 2. E214G, R222Q and D344N are common to both 2B and 2C * representative of recent subgroups

12 Table 4. Antigenic analyses of influenza A H3N2 viruses (Turkey RBCs) Haemagglutination inhibition titre 1 Post infection ferret sera Viruses Collection Passage A/Wis A/Trieste A/Wis A/Bris A/Uru A/JHB A/Eng A/Bris Date History 67/05 25c/07 3/07 10/07 716/07 15/08 394/08 24/08 F18/08 F5/07 F15/07 F29/08 F26/08 F22/08 F32/08 F3/09 REFERENCE VIRUSES A/Wisconsin/67/ /08/2005 SpfCk3E3 \ A/Trieste/25c/2007 Jan-07 MDCKx \2 < A/Wisconsin/3/ /01/2007 Ex \ A/Brisbane/10/ /02/2007 E2 \ A/Uruguay/716/ /06/2007 spfck1, E3 \ A/Johannesburg/15/ /06/2008 MDCKx \2 < A/England/394/ /09/2008 MDCK2 \ A/Brisbane/24/ /06/2008 E5 \ TEST VIRUSES A/Madagascar/3226/ /09/2008 MDCK1 \1 < ND A/Netherlands/379/ /09/2008 MK2 \1 < < < 40 ND A/Norway/2118/ /11/2008 MDCK1 \1 < ND 80 ND A/Thuringen/76/ /11/ Pa(RKI) \1 < ND 80 ND A/Luxembourg/1325/ /12/2008 MDCKx \ ND A/England/561/ /12/2008 MDCK P1 \1 < ND A/England/572/ /12/2008 MDCK P1 \1 < < 40 ND A/Finland/286/ /12/2008 MDCK2 \1 < ND A/England/ / /12/2008 SIAT P2 \1 < ND A/Paris/130/ /10/2008 C2 \ A/Denmark/191/ /11/2008 MDCK3 \ A/Toulouse/1317/08 14/11/2008 P3MDCK \1 < A/Egypt/59/ /11/2008 C1 \1 < A/Hong Kong/3197/ /11/2008 MDCK2 \ A/Sweden/6/08 08/12/2008 MDCK2 \ A/Norway/2308/ /12/2008 MDCK1 \ A/England/669/ /12/2008 MDCK P1 \ A/Norway/1/ /12/2008 MDCK1 \ A/Kanagawa/65/2008 unknown MDCK2+2 \1 < A/Taiwan/471/2008 unknown E3+2 \ A/Stockholm/26/ /11/2008 MDCK1 \ A/Norway/2264/ /12/2008 MDCK1 \1 < A/Baden-Württemburg/199/ /12/2008 MDCK4 \2 < A/Bayern/47/ /12/2008 MDCK5 \1 < A/Niedersachsen/107/ /12/2008 MDCK2 \1 < A/Sweden/9/ /12/2008 MDCK2 \ A/Baden-Württemburg/196/ /12/2008 MDCK3 \1 < A/Niedersachsen/110/ /12/2008 MDCK4 \ A/Berlin/62/ /12/2008 MDCK4 \1 < A/Firenze/24/08 22/12/2008 I MDCK \1 < A/Firenze/26/08 22/12/2008 I MDCK \1 < A/Switzerland/ / /12/2008 MDCK1 \1 < A/Switzerland/ / /12/2008 MDCK2 \1 < A/Parma/20/09 30/12/2008 MDCK1 \1 < A/Latvia/1-34/ /01/2009 MDCK1 \2 < A/Bremen/1/ /01/2008 MDCK3 \ A/Slovenia/51/ /01/2009 MDCKx \1 < A/Slovenia/55/ /01/2009 MDCKx \1 < A/Parma/22/09 10/01/2009 MDCK1 \1 < A/Parma/17/09 12/01/2009 MDCK1 \1 < A/Lisboa/04/ /12/2008 MDCK2 \2 < A/Cluj/34/ /01/2009 MDCK3 \2 < A/Barcelona/321/ /12/2008 MDCK2 \2 < A/Barcelona/328/ /12/2008 MDCK2 \2 < A/Algeria/G208/ /12/2008 Cx \1 < A/Bucuresti/1816/ /11/2008 MDCK 3 \1 < A/Lasi/22/ /01/2009 MDCK 3 \1 < A/Cluj/35/ /01/2009 MDCK 3 \1 < A/Bucuresti/54/ /01/2009 MDCK 2 \1 < A/Israel/9/09 10/01/2009 MDCKx \1 < A/Israel/13/09 17/01/2009 MDCKx \1 < < A/Barcelona/329/ /12/2008 MDCK2 \1 < < A/Barcelona/V359A/ /01/2009 MDCK1 \1 < A/Algeria/G216/ /12/2008 Cx \1 < A/Algeria/G217/ /12/2008 Cx \1 < A/Firenze/14/08 27/11/2008 I MDCK \2 < A/Bratislava/148/ /01/2009 1\ A/Bratislava/151/ /01/2009 1\ A/Belgium/G318/ /01/2009 MDCK1 \3 < A/Belgium/G344/ /01/2009 MDCK1 \3 < < = <40; ND = not done Also tested by neutralisation Vaccine strains

13 Table 5. Antigenic analyses of influenza A H3N2 viruses (Guinea Pig RBCs) Haemagglutination inhibition titre 1 Post infection ferret sera Viruses Collection Passage A/Wis A/Trieste A/Wis A/Bris A/Uru A/JHB A/Eng A/Bris Date History 67/05 25c/07 3/07 10/07 716/07 15/08 394/08 24/08 F18/08 F5/07 F15/07 F29/08 F26/08 F22/08 F32/08 F3/09 REFERENCE VIRUSES A/Wisconsin/67/ /08/2005 SpfCk3E3 \ A/Trieste/25c/2007 Jan-07 MDCKx \ A/Wisconsin/3/ /01/2007 Ex \ A/Brisbane/10/ /02/2007 E2 \ A/Uruguay/716/ /06/2007 spfck1, E3 \ A/Johannesburg/15/ /06/2008 MDCKx \ A/England/394/ /09/2008 MDCK2 \ A/Brisbane/24/ /06/2008 E5 \ TEST VIRUSES A/England/392/ /09/2008 C1 \ ND A/England/394/ /09/2008 C2 \ ND A/England/395/ /09/2008 C2 \ ND A/Mauritius/L1/ /11/2008 Cx \ ND A/Paris/458/ /12/2008 C1 \ ND A/Singapore/N593/ /09/2008 Cx \ A/Sweden/3/08 15/10/2008 MDCK2 \ A/England/687/ /12/2008 MDCK P1 \ A/Switzerland/ / /12/2008 MDCK2 \ A/Seoul/4436/ /12/2008 MDCK-P2 \ A/Slovenia/60/ /01/2009 MDCKx \ A/Slovenia/51/ /01/2009 MDCKx \ A/Baden-Württemburg/199/ /12/2008 C4 \ A/Parma/17/09 12/01/2009 I MDCK \ A/Switzerland/ / /12/2008 cs \ A/Leon/5/ /01/2009 MDCK1 \ A/Clermont-F./1304/08 17/11/2008 P2MDCK \ ND A/Poitiers/1341/08 04/11/2008 Px+P1MDCK \ ND A/Paris/458/ /12/2008 C1 \ ND A/England/2/ /12/2008 SIAT P1 \ ND A/Norway/14/ /01/2009 MDCK1 \ ND A/Thüringen/82/ /12/2008 C1 \ ND A/Baden-Württemburg/194/ /12/2008 C3 \ ND A/Torino/13/08 03/12/2008 MDCK1 \ A/Roma-ISS/3/09 12/01/2009 MDCK1 \ A/Bratislava/144/ /01/2009 MDCK1 \ A/Algeria/G155/ /12/2008 Cx \ A/Switzerland/ / /01/2009 cs \ A/Switzerland/ / /01/2009 cs \ A/Tanger/536/09 13/01/2009 Cx \ A/Firenze/25/08 22/12/2008 MDCK1 \ A/Roma-ISS/9/09 20/01/2009 MDCK1 \ A/Salamanca/10/ /01/2009 MDCK1 \ A/Bratislava/96/ /12/2008 MDCK1 \ A/Meknes/518/09 13/01/2009 Cx \ A/Tanger/531/09 05/01/2009 Cx \ A/Soria/3/ /01/2009 MDCK1 \ A/Burgos/4/ /01/2009 MDCK1 \ A/Fes/521/09 31/12/2008 Cx \ A/Tanger/533/09 20/12/2008 Cx \ A/Torino/14/08 12/12/2008 MDCK1 \ A/Roma-ISS/7/09 13/01/2009 MDCK1 \ A/Roma/18/08 10/12/2008 MDCK1 \ A/Athens/14/ /01/2009 Cx \ A/Athens/15/ /01/2009 Cx \ A/Algeria/G202/ /12/2008 Cx \ A/Genoa/09/ /01/2009 MDCK1 \ A/Genoa/10/ /12/2009 MDCK1 \ A/Belgium/SP07/ /12/2008 P1? \ A/Belgium/SP08/ /12/2008 P1? \ A/Algeria/G120/ /11/2008 Cx \ A/Roma-ISS/2/09 12/01/2009 MDCK1 \ A/Parma/46/08 16/12/2008 MDCK1 \ A/Parma/52/08 31/12/2008 MDCK1 \ A/Valladolid/12/ /01/2009 MDCK1 \ A/Salamanca9/ /01/2009 MDCK1 \ A/Barcelona/V258A/ /01/2009 1\ A/Barcelona/V266A/ /01/2009 1\ A/Lisboa/43/ /12/2008 MDCK 2 \ A/Lisboa/01/ /01/2009 MDCK 2 \ A/León/13/ /01/2009 MDCK 1 \ A/Valladolid/14/ /01/2009 MDCK 1 \ A/Prague/63/ /01/2009 MDCK2 \ < = <40; ND = not done Also tested by neutralisation Vaccine strains

14 Table 6. Antigenic analysis of influenza A H3N2 viruses (Neutralisation) Neutralisation titre 1 Post-infection ferret sera 2 Viruses Collection Passage A/Well A/Bris A/Uru A/JHB A/Eng A/Bris Date History 1/04 10/07 716/07 15/08 394/08 24/08 F13/04 F18/07 F3/07 F22/08 F32/08 F3/09 REFERENCE VIRUSES A/Wellington/1/ /01/2004 E3 \ A/Brisbane/10/ /02/2007 E2 \ A/Uruguay/716/ /06/2007 spfck1, E3 \ A/Johannesburg/15/ /06/2008 MDCKx \ ND A/England/394/ /09/2008 TC P2 \ A/Brisbane/24/08 23/06/2008 E ND TEST VIRUSES A/England/561/ /12/2008 MDCK P1 \ ND A/Finland/286/ /12/2008 MDCK2 \ ND A/England/ / /12/2008 SIAT P2 \ ND A/Madagascar/3226/ /09/2008 MDCK1 \ ND A/Netherland/379/ /09/2008 MK2 \ ND A/Luxembourg/1325/2008 unknown MDCKx \ ND A/England/572/ /12/2008 MDCK P1 \ ND A/England/392/ /09/2008 TC P1 \ ND A/England/394/ /09/2008 TC P2 \ ND A/England/395/ /09/2008 TC P2 \ ND A/Thuringen/76/ /11/2008 Cx \ ND A/Norway/2118/ /11/2008 MDCK \ ND A/Mauritius/L1/ /11/2008 Cx \ ND A/Paris/130/ /10/2008 C2 \ ND A/Egypt/59/ /11/2008 C1 \ ND A/Toulouse/1317/ /11/2008 P3MDCK \ ND A/Norway/2308/ /12/2008 MDCK1 \ ND A/Denmark/191/ /11/2008 MDCK3 \ ND A/Sweden/6/ /12/2008 MDCK2 \ ND A/Paris/458/2008 1/12/2008 C1 \ ND A/HongKong/3197/ /11/2008 MDCK2 \ ND A/England/669/ /12/2008 MDCK P1 \ ND A/Norway/1/ /12/2008 MDCK1 \ ND Based on 50% plaque reduction compared to serum negative controls 2 RDE treated ND = not done Agglutinated guinea pig RBCs only Vaccine strains

15 Figure 4. Phylogenetic comparison of influenza H3N2 HA genes Vaccine strain Reference strains Isolates November 2008 December 2008 January 2009 G50E K140I K173Q h3england57208d h3denmark20008ded h3slovenia6009j h3england69908d h3burgos3308d h3switzerland d h3finland28908fnn h3england d h3thuringen7608n h3finland28608fnd h3austria auj h3norway1209nwj h3sweden708sed h3norway231408d h3slovenia5509j h3england68708d h3switzerland chj h3belgiumg25409j h3trieste2809itj h3norway223508d h3england69508d h3stockholm2708sed h3sweden508d h3montana0208cdcd h3belgiumg30809j h3genoa0909itj h3badenw19408d h3paris81708d h3switzerland d h3paris77708d h3denmark19108n h3latvia14109j h3norway207908nwn h3berlin6308ged h3austria auj h3norway1409j h3stockholm2508o h3bremen708gen h3badenw19608d h3austria auj h3switzerland chj h3nordrheinw021208ged h3paris30308frn h3norway109j h3norway212608n h3parma5308d h3latvia d h3trieste2609itj h3firenze2408d h3switzerland chj h3yamaguchi3508japd A/England/394/2008 h3denmark19808ded A/Brisbane/24/2008 h3wisconsin1808cdcd h3paris26308frn h3lisbon3408pgd h3genoa0509itj h3luxembourg137808d h3poitiers134108n h3singaporen59308s h3seoul443608d h3bayern4408ged h3lisbon3308pgd h3tunisia72309j h3sweden908sed h3genoa1009itj A/Johannesburg/15/2008 h3leon509j h3singaporen36208a h3paris00308frg A/Uruguay/716/2007 A/Trieste/25/2007 A/Wisconsin/3/2007 A/Brisbane/10/2007 A/Finland/9/2008 h3nepal92106 A/Wisconsin/67/2005 calif7e04cdcs h3victoria50009ausj 10 nucleotide substitutions

16 Figure 5. Phylogenetic comparison of influenza H3N2 neuraminidase genes Vaccine strain Reference strains Isolates November 2008 December 2008 January 2009 V194I N387K Y310H S372L N387K D147N I215V H150R P386H I464L h3england66908n2d h3sweden508n2d h3finland28908n2fnn h3finland28608n2fnd h3thuringen7608n2n h3belgiumg30809n2j h3england68708n2d h3belgiumg25409n2j h3england69508n2d h3paris28808n2frn h3england57208n2d h3england n2d h3netherlands37908n2s h3sweden708n2sed h3switzerland n2d h3england65208n2d h3badenw19408n2d h3montana0208n2cdcd h3slovenia6009n2j h3denmark20008n2ded h3denmark19608n2ded h3england69908n2d h3norway231408n2d h3norway223508n2d h3stockholm2708n2sed h3slovenia5509n2j h3denmark19308n2ded h3denmark19808n2ded h3singaporen42508n2y h3parma5308n2d h3luxembourg137808n2d h3latvia n2d h3lisbon3408n2d h3seoul443608n2d h3stockholm2608n2sen h3singaporen59308n2s A/England/394/2008 h3norway109n2 h3norway1409n2j h3sweden408n2sen h3paris77708n2d h3paris81708n2d h3switzerland n2d h3denmark19108n2den h3stockholm2408n2seo h3latvia14109n2j h3paris30308n2frn h3badenw19608n2d h3paris26308n2frn h3poitiers134108n2n h3laplata n2l h3wisconsin1808n2cdcd h3mauritiusl308mp3n2 h3norway212608n2n h3norway214508n2nwd h3firenze2408n2d h3sweden908n2sed h3stockholm2508n2seo h3stockholm2508n2o h3parma3908n2 h3norway207908n2nwn h3norway216308n2nwn h3kentucky108n2cdcs A/Brisbane/10/2007 A/Wisconsin/3/2007 A/Trieste/25/2007 A/Uruguay/716/2007 h3nepal92106n2 h3prague307n2 A/Wisconsin/67/2005 h3henan14707n2cdcj n2california704cdcs A/Brisbane/24/2008 A/Johannesburg/15/2008 h3leon509n2j h3singaporen36208n2a A/Finland/9/2007 h3paris00308n2frg h3victoria50009n2ausj h3hongkong186808n2cdcl 10 nucleotide substitutions

17 Table 7. Antigenic analyses of influenza B viruses (Victoria lineage) Haemagglutination inhibition titre 1 Post infection ferret sera Viruses Collection Passage B/Shan 2 B/Shan B/Bris B/HK B/Mal B/Vic B/Eng B/Bris B/Bris date History 7/97 7/97 32/02 45/ /04 304/06 393/08 60/08 33/08 F12/02 F2/03 F15/05 F28/05 F16/06 F31/08 F2/09 AUS F1235 REFERENCE VIRUSES B/Shandong/7/97 Ex B/Brisbane/32/ /07/2002 E2 \ B/HK/45/ /02/2005 MDCK2 \ < < B/Malaysia/2506/ /12/2004 E3 \ B/Victoria/304/ /06/2006 E2 \ B/England/393/ /08/2008 E1 \ < B/Brisbane/33/ /07/2008 E3 \ < B/Brisbane/60/ /08/2008 E4 \ < TEST VIRUSES B/Hong Kong/1347/ /09/2008 MDCK2 \ < < 320 < < < < 40 B/Hong Kong/1373/ /10/2008 MDCK2 \ < < 160 < < < < 40 B/Agadir/235/09 11/12/2008 Cx \ < < 160 < < < < 40 B/Tanger/522/09 19/12/2008 Cx \ < < < < 160 B/Tanger/438/09 30/12/2008 Cx \ < < 160 < < < < 80 B/England/457/08 31/10/2008 PLC / Egg P3 \ < B/Texas/26/ /11/2008 E3 \ < B/Baden Wurttemberg/158/ /10/ Pa(RKI) \1 640 < < < < < < B/Bayern/36/ /10/2008 MDCK2 \1 640 < < < < < < ND 1280 B/Singapore/14/ /11/2008 MDCK1 \ < < < < < < < 40 B/Belgium/G086/ /11/2008 MDCK1 \ < < < 40 < < B/England/558/ /12/2008 SIAT P1 \1 640 < < < < < < B/Bayern/40/ /12/2008 MDCK2 \1 640 < < < < < < B/Bayern/39/ /12/2008 MDCK2 \1 640 < < < < < < < 40 B/Niedersachsen/183/ /12/2008 MDCK2 \1 640 < < < < < < B/England/711/ /12/2008 MDCK P1 \1 640 < < < < < < B/Berlin/42/ /12/2008 MDCK2 \1 640 < < < < < < B/Switzerland/ / /01/2009 cs \4 640 < < < < < < B/Barcelona/98/ /11/2008 0\1 320 < < < < < < B/Barcelona/99/ /11/2008 0\1 640 < < < < < < B/Barcelona/V362A/ /01/2009 1\1 640 < < < < < < B/Israel/2/09 11/01/2009 MDCKx \1 320 < < < < < < B/Israel/3/09 17/01/2009 MDCKx \1 320 < < < < < < B/Bratislava/108/ /01/2009 1\1 320 < < < 40 < B/Volos/16/ /01/2009 Cx \3 320 < < < 10 < < 80 ND B/Volos/68/ /01/2009 Cx \3 160 < < < 10 < < 80 ND B/Athens/84/ /01/2009 Cx \3 160 < < < 10 < < 80 ND 1. < = <10; 2. hyperimmune sheep serum Vaccine strain ND = not done

18 Figure 6. Phylogenetic comparison of influenza B HA genes (Victoria lineage) Vaccine strain Reference strains Isolates October 2008 November 2008 December 2008 victoria287 beij187 L58P K48E K80R K129N hongkong7694 hong7096 hk33001a N165K S172P N75K B/Brisbane/33/2008 B/England/393/2008 bengland39308tcg bsakai1408japo bsakai1208japo bengland45708o bsakai3208japn bengland45208n byokohama708japd bsydney20208ausl btexas2408cdco btexas3408cdcd btexas2608cdcn btexas2308cdco bbelgiumg08608han bosaka1508japo bkobe4708japn bnara808japd bhyogo908japd bbayern4008had bbayern3608o bbayern3908had B/Brisbane/60/2008 bhawaii0608cdco bengland55808d bbadenw15808o bflorida2508cdcd bslovenia25208haj bdnipro15608haf bagadir23508had btanger43809had begypt8007d bvalladolid1108haj bparma4508haf bparis023107o bdakar1307y V225I V146I B/Victoria/304/2006 bsingapore1408n bmauritius46807l bpennsyl507cdc bpiura000608db bastrakhan8807u bbadenwurt107j bmauritius40307u bmadagascar120907l bjohannesburg4308u bjohannesburg808a bjohannesburg15707l bhongkong132807d bhongkong99808m bhongkong133207d bhongkong9008cdcf btochigi1508japo btochigi1208japo btochigi1308japo btianjin3208cdcj bhongkong134708d balgeriag2108 btrieste808haj bjiangxi1308cdcj bfujiangulou127208cdcm bflorida1608cdcd bwisconsin2708cdcd bbuenosaires hal balaska108cdcj bbrazil293708ausl B/Malaysia/2506/2004 bhk85906l B/Hong Kong/45/2005 bguayaquil982908db bpnguinea308ausa B/Brisbane/32/2002 tehran8002f beijing24396 B/Shandong/7/97 Clade 2 Clade 1 10 nucleotide substitutions

19 Figure 7. Phylogenetic comparison of influeza B neuraminidase genes Vaccine strain Reference strains Isolates October 2008 November 2008 Dec Jan 2009 P42Q K125T T68A R186K N340D D463N A465T Q42R bnorway137808nau bghanamh4108nas bnghanafs42808d blatvia424608naa B/Valladolid/18/2008 bnghanafs38508n bghanamh3608nag bghanangpk0108nas bmauritius37108nau bmauritius38708nal blyon106208nau bjohannesb32108nal brzeseow1108nam bdakar608nag bghanafs25008nas bnbayern3708n bfinland24808nam bswitzerland nad bthuringen4308nam bdenmark9708nam bdakar308nal bnhongkong137208s B/England/145/2008 bnalgeriag7608 bnnetherlands37508s biceland8108nam begypt13309naj bhk120808nal bnniedersachsen18208o bntexas3708cdcd bnwashington0408cdcd B/Bangladesh333307cdcg B/Egypt/144/2005 bmadagascar192708naf B/Florida/7/2004 B/Florida/4/2006 bbratislava41108naa blaplata nau blyon99908naa bpalencia3008naa bmendozar53008na bberlin3308nam bnorway120908nay bhongkong111908naa B/Brisbane/3/2007 bulan-ude508naa bnsendaih11407jap bcapetown3008nam bcapetown33108nal bbayern3908nad bbayern4008nad bnbayern3608o bnengland45708o bnengland45208n K125N E320D E404K Q61H D329N I204V A358E N220K S41P P42S V271I D463N B/England/393/2008 B/Brisbane/33/2008 bntexas3408cdcd bbelgiumg08609nan bntexas2608cdcn Clade 3 Clade 1 Clade 2 B/Brisbane/60/2008 bnengland55808d bnflorida2508cdcd bslovenia25208naj bdnipro15608naf b235agadir09nad btanger43809nad bvalladolid1108naj bjohannesburg4308nau bjohannesburg808naa B/Victoria/304/2006 bnsingapore1408n bparma4508naf bhongkong99808nam bnhongkong9008cdcf bnbrazil293708ausl bnwisconsin2708cdcd D342G M375K balaska bnalgeriag2108 bhk124608nal bnhongkong134708s bnfujiangulou127208cdcm B/Malaysia/2506/2004 B/Hong Kong/45/2005 B/Shandong/7/97 Yamagata-lineage HA Clade 2A Clade 2B Clade 1 Victoria-lineage HA 10 nucleotide substitutions

20 Table 8. Antigenic analyses of influenza B viruses (Yamagata lineage) Haemagglutination inhibition titre 1 Post infection ferret sera Viruses Collection Passage B/Fl 2 B/Eg B/Fl B/Bris B/Barc B/Eng B/Vall B/Ban date History 4/06 144/05 4/06 3/07 143/08 145/08 18/ /07 Sh 478 F3/07 F19/07 F24/07 F11/08 F9/08 F10/08 F21/08 REFERENCE VIRUSES B/Egypt/144/ /05/2005 E2 \ B/Florida/4/ /12/2006 E2 \ B/Brisbane/3/ /09/2007 E2 \ B/Barcelona/143/ /01/2008 MDCK1 \ B/England/145/2008 Ex \ < B/Valladolid/18/ /02/2008 MDCK1 \ B/Bangladesh/3333/ /08/2007 E4 \ TEST VIRUSES B/Dakar/06/ /08/2008 MDCK2 / B/Hong Kong/1355/ /09/2008 MDCK2 \ B/Ghana/MH-41/ /09/2008 MDCKx / B/Hong Kong/1372/ /09/2008 MDCK2 \ B/Ghana/FS301/ /10/2008 MDCK2 \ B/Hong Kong/1374/ /10/2008 MDCK2 \ B/Ghana/FS347/ /10/2008 MDCK2 \ B/Ghana/FS351/ /10/2008 MDCK2 \ B/Paris/236/ /11/2008 C2 \ < < B/Ghana/FS435/ /12/2008 MDCK2 \ B/Switzerland/ / /12/2008 MDCK1 \ B/Egypt/133/ /01/2009 Ex \1 160 < < < < 20 < 10 B/Belgium/G212/08 24/12/2007 MDCKx \ B/Switzerland/ / /12/2008 MDCK1 \ B/Bayern/37/ /11/ Pa \ B/Cameroon/08-177/ /11/2008 MDCKx \ ND B/Cameroon/08-229/ /12/2008 MDCKx \ ND B/Barcelona/51/ /10/2008 0\ < ND < < < B/Norway/2054/ /11/2008 MDCK1 \ B/Pavia/3/08 03/11/2008 I MDCK \ < = <10; 2. hyperimmune sheep serum ND = not done Vaccine strains

21 Figure 8. Phylogenetic comparison of influenza B HA genes (Yamagata lineage) Vaccine strains Reference strains Isolates October 2008 November 2008 Dec Jan 2009 P108A S150I N165Y G229D R48K bhk120808hau blyon106208u bhk126408hal bhongkong137208s bdenmark9708m begypt13309haj bnetherlands37508s bmie09japj bfinland24808m bmoscow208haf bengland35508 B/England/145/2008 balaska0508cdcn bnorway54208f bgeneva970708f bakita208japn bpavia308ha brzeszow1108m balgeriag7608 bghanamh3608g bghanamh4108s bghanangpk0108s bnorway205408nwn bsalta171008haj bnorway137808hau btexas3708cdcd bmichigan1108cdcn bghanafs36508ha B/Barcelona/143/2008 blatvia424608a B/Valladolid/18/2008 bmures64008f bghanafs38508n bmauritius37108u bghanafs42708had bmauritius38708l bdakar608hag bdakar308hal bsingaporen41608hay bghanafs25008s biceland8108m bbayern3708n bnis49708 bthuringen4308m bjohannesb32108hal bwashington0408cdcd bireland342108m bswitzerland had B/Bangladesh/3333/2007 B/Brisbane/3/2007 bnorway120908hay balaska0308cdcn bhongkong111908a bulanude508a bsingaporeen07808han bsingaporen43108hay bcapetown3008m bcapetown33108hal bsendaih11407ejap bdenmark8508a B/Egypt/144/2005 bmadagascar192708f blyon99908a bbolivia48808cdcn G229S N202S B/Florida/7/2004 K271R bbsas u bkawasaki208japn bniigata116408japd bberlin3308m btaiwan39908japn bmendozar53008ha bbratislava41108haa bgeneva bluxembourg94608ha blisbon1208haf blaplata u B/Florida/4/2006 bbraila70108m bpavia208its bpalencia3008a Clade 2 Clade 3 Clade 1 10 nucleotide substitutions

22 Table 9. Amino acid changes characteristic of recent influenza B sequence variants HA Lineage Representative strain HA NA Clade Changes Clade Changes Yamagata 1 B/Florida/4/ G229S 1 T68A K271R K125T D478E R186K N340D B/Brisbane/3/ R48K 2 T68A P108A K125T R186K I248V N340D B/England/145/ S150I 3 Q42R N165Y D463N G229D A465T Victoria 2 B/Hong Kong/1347/ D342G M375K B/Brazil/2937/ B P41S S42P Q61H K125N I271V E320D E404K N463D B/Brisbane/60/ N75K 2A P41S V146I S42P N165K K125N S172P N220K (L58P) I271V (V225I) E320D A358E E404K N463D (I204V) (D329N) 1. Relative to B/Florida/7/ Relative to B/Malaysia/2506/04.