[Source: European Centre for Disease Prevention and Control (ECDC), full PDF document. (LINK). Edited.]
Influenza virus characterisation, Summary Europe, July 2012
During the 2012–13 season, A(H1N1)pdm09, A(H3N2) and B/Victoria- and B/Yamagata-lineage influenza viruses have been detected in ECDC-affiliated countries. The relative prevalences varied between countries.
- Type A and type B viruses have continued to co-circulate in similar proportions.
- A(H1N1)pdm09 viruses have been detected at comparable levels to A(H3N2) viruses.
- A(H1N1)pdm09 viruses continued to show genetic drift from the vaccine virus, A/California/07/2009, but the vast majority remained antigenically similar to it.
- The vast majority of A(H3N2) viruses have been antigenically and genetically similar to cell-propagated A/Victoria/361/2011, the prototype vaccine virus for the 2012–13 influenza season.
- Viruses of the B/Yamagata lineage predominated over those of the B/Victoria lineage.
- B/Victoria lineage viruses were antigenically similar to cell-propagated reference viruses of the B/Brisbane/60/2008 genetic clade.
- Recent B/Yamagata-lineage viruses fell into two antigenically distinguishable genetic clades: clade 2, represented by B/Estonia/55669/2012, and clade 3, represented by B/Wisconsin/1/2010 (the recommended vaccine component for the 2012–13 influenza season).
Viruses collected between 1 December 2012 and 31 May 2013, spanning the 2012–13 season, were received from 23 countries in the EU/EEA region at the MRC National Institute for Medical Research, WHO Collaborating Centre for Reference and Research on Influenza. A summary of specimens received is shown in Table 1.
The proportions of influenza type A (61%) and type B (39%) viruses received were similar. For type A, H1N1pdm09 viruses were received in greater numbers than H3N2 viruses (ratio 2:1). Among influenza B receipts, viruses of the B/Yamagata and B/Victoria lineages were received at a ratio of 4:1.
Influenza A(H1N1)pdm 09 virus analyses
The results of HI assays carried out on influenza A(H1N1)pdm09 viruses since the April report  are shown in Table 2. All test viruses continued to show good reactivity with post-infection ferret antisera raised against the panel of reference viruses, including antiserum raised against the vaccine virus, A/California/7/2009, with this antiserum recognising the test viruses shown at titres within fourfold of its recognition of the homologous virus. As described previously , antiserum raised against A/Christchurch/16/2010, a virus from a genetic group not seemingly in circulation at present (group 4), reacted less well than the other antisera with the test viruses: this ferret antiserum reacted with approximately 30% (11/29) of the test viruses, with titres reduced eightfold or greater compared with the titre of the antiserum with the homologous virus.
Antisera raised against several of the reference strains reacted poorly with a single virus, A/Plzen/18/2013, showing eightfold or greater reductions in titre compared to the titres of the antisera with their homologous antigens; HA gene sequencing of this virus showed that it carried a polymorphism at residue 155 (E>G) of HA1. Amino acid substitution or polymorphism in this region of HA1 can affect the antigenicity of the virus and commonly emerges during propagation of viruses in cell culture.
Phylogenetic analysis of the HA gene of representative viruses (Figure 1) shows that the H1N1 viruses from EU/EEA countries collected during the 2012–13 season cluster within genetic groups 6 and 7, with viruses belonging to group 6 predominating. HA gene sequencing was performed on 19 test viruses and the genetic groups to which they belong are shown in Table 2; all but two were in genetic group 6.
Influenza A(H3N2) virus analyses
Influenza A(H3N2) viruses continue to be difficult to characterise antigenically by HI assay due to variable agglutination of red blood cells from guinea pigs, turkeys and humans as described before . The change in agglutination of red blood cells is associated with a reduced avidity of H3N2 viruses for the sialic acid receptor on the surface of the cell . Antigenic analyses of recently collected viruses conducted since the April report  are shown in Table 3. HI assays were carried out using guinea pig red blood cells in the presence of 20nM oseltamivir, added to circumvent the NA-mediated binding of H3N2 viruses to the red blood cells . The test viruses reacted poorly with post-infection ferret antiserum raised against the egg-propagated vaccine virus for 2012–13, A/Victoria/361/2011, compared with the titre against the homologous virus.
Generally, the test viruses also reacted poorly with antisera raised against other reference viruses and previous vaccine viruses propagated in eggs (A/Perth/16/2009, A/Victoria/208/2009, A/Iowa/19/2010 and A/Hawaii/22/2012). However, overall the panel of test viruses showed better reactivity with antiserum raised against egg-propagated A/Texas/50/2012 (the H3N2 vaccine virus recommendation for the northern hemisphere 2013–14 ) compared with the titre of the antiserum with the homologous virus than they did against other egg-propagated viruses. In Table 3, antiserum raised against A/Texas/50/2012 recognised 7 out of 25 of the test viruses with titres within fourfold of the titre to the homologous virus. A/Texas/50/2012 is an A(H3N2) virus antigenically like the cell-propagated prototype virus A/Victoria/361/2011.
The test viruses reacted well with antisera raised against reference viruses exclusively propagated in MDCK cells, and/or the derivative MDCK-SIAT-1 cells when compared to the titres with the homologous viruses. These antisera were raised against cell-propagated virus isolates of A/Victoria/361/2011, A/Alabama/5/2010, A/Stockholm/18/2011, A/Berlin/93/2011 and A/Athens/112/2012.
Phylogenetic analysis of the HA gene sequences of representative viruses is shown in Figure 2. Viruses from EU/EEA countries collected since 1 December 2012 have HA genes that fall predominantly into genetic group 3C. Viruses carrying HA genes falling into group 3A and 3B (described in previous reports), 5 (e.g. A/Plzen/22/2013) and 6 (e.g. A/Lisboa/SU91/2012) have also been isolated since December 2012.
The amino acid substitutions in HA1/HA2 associated with these groupings of recently collected viruses are:
- Group 3 viruses:
- N145S and V223I, with viruses in Groups 3B and 3C also carrying A198S and N312S
- Group 3C:
- S45N (resulting in gain of a potential glycosylation site) and T48I, e.g. the prototype vaccine virus A/Victoria/361/2011; the great majority of viruses also carry the substitutions Q33R and N278K (e.g. A/Berlin/93/2011); an emerging subgroup also carries the substitutions T128A (resulting in the loss of a potential glycosylation site) and R142G
- Group 3B:
- Group 3A:
- N144D, D158N
- Group 5 viruses:
- D53N, Y94H, I230V and E280A (e.g. A/Alabama/05/2010), often in combination with K2E, N8D (resulting in the loss of a potential glycosylation site) and S124N
- Group 6 viruses:
- D53N, Y94H, S199A, I230V and E280A (e.g. A/Iowa/19/2010).
There is no evidence for antigenic change associated with any of the genetic groups or emerging subgroups, including the emerging subgroup in group 3C that carries substitutions in HA1 at amino acid residues 128 and 142.
Influenza B virus analyses
Table 4 shows the results of antigenic analyses for viruses of the B/Victoria lineage performed since the April report . All test viruses were from Slovenia. Compared with the titre against the homologous virus in HI assays all test viruses showed low reactivity with post-infection ferret antiserum raised against the egg-propagated virus B/Brisbane/60/2008, a component of trivalent vaccines for the 2010–11 season and a recommended component of quadrivalent vaccines  for the 2013–14 northern hemisphere influenza season. The test viruses showed a similarly reduced reactivity with antisera raised against other reference viruses propagated in hens' eggs: B/England/393/2008, B/Malta/636714/2011 and B/Johannesburg/3964/2012. The test viruses reacted better with antisera raised against reference viruses genetically closely related to B/Brisbane/60/2008 but propagated in cells; these post-infection ferret antisera were raised against B/Paris/1762/2008, B/Hong Kong/514/2009, B/Odessa/3886/2010 and B/Formosa/V2367/2012.
Phylogenetic analysis of the HA genes of representative B/Victoria lineage viruses is shown in Figure 3. All the viruses received with collection dates in 2013 from EU/EEA laboratories carried HA genes that fell into genetic clade 1A. The amino acid substitution associated with the separation of clade 1 into clades 1A, 1B and L58P has no apparent effect on antigenicity. The HAs of recent viruses show few amino acid substitutions compared with B/Brisbane/60/2008.
Table 5 shows the results of HI analyses of B/Yamagata lineage viruses tested since the April report . The genetic clade into which sequenced HA genes of test viruses fall is indicated.
All 16 test viruses showed good reactivity (within fourfold of the homologous titre) with antisera raised against the egg-propagated vaccine virus recommended for the northern hemisphere winter 2013–14 influenza season  B/Massachusetts/02/2012. Antiserum raised against egg-propagated B/Wisconsin/1/2010 – the virus used in the vaccine for 2012–13 – also showed reactivity within fourfold of the titre against the homologous virus for the majority of test viruses.
Antisera raised against cell-propagated viruses, whether of clade 2 or clade 3, showed good reactivity (within fourfold of the homologous titre) against the test viruses. Nine of the test viruses had been genetically characterised at the time of preparation of this report, with six falling into genetic group 2 and three into group 3.
Figure 4 shows a phylogenetic analysis of the HA genes of representative B/Yamagata lineage viruses. The analysis shows that the HA genes of recent viruses continue to fall into two genetic clades: clade 3 (represented by the vaccine virus B/Wisconsin/1/2010 and reference viruses B/Stockholm/12/2011 and B/Novosibirsk/1/2012) and clade 2 (represented by the reference viruses B/Brisbane/3/2007, B/Estonia/55669/2011, B/Hong Kong/3577/2012 and the 2013–14 vaccine virus B/Massachusetts/02/2012). The two clades are differentiated by substitutions at HA1 residues 48, 108, 150, 165, 181 and 229. The HA genes of viruses of clade 2 encode K48, A108, S150, N165, A181 and G229; the HA genes of viruses in clade 3 encode R48, P108, I150, Y165, T181 and D229.
The proportion of viruses received with HA genes that fall into clade 2 has continued to increase over the number with HA genes falling into clade 3.
Influenza A(H7N9) virus
On 1 April 2013, the WHO Global Alert and Response  reported that the China Health and Family Planning Commission notified the World Health Organization (WHO) of three cases of human infection with influenza A(H7N9). The cases were confirmed by laboratory testing on 29 March by the Chinese CDC. A description of the characteristics of H7N9 viruses can be found on the WHO website . WHO is updating information on the outbreak regularly and ECDC is posting epidemiological updates . A Rapid Risk Assessment  for these A(H7N9) viruses has been carried out and posted by ECDC on 3 April 2013 and an updated risk assessment has been posted by WHO. As of 30 May 2013, WHO reported  132 laboratory confirmed cases and 37 associated fatalities.
A description of results generated by the WHO Collaborating Centre for Reference and Research on Influenza at the MRC National Institute for Medical Research in London, and evaluated at the WHO Vaccine Composition Meetings held in Beijing, China, on 17–19 September 2012 and at WHO Geneva on 18–20 February 2013, can be found at: http://www.nimr.mrc.ac.uk/documents/about/Interim_Report_September_2012_2.pdf http://www.nimr.mrc.ac.uk/documents/about/Interim_Report_February 2013.pdf 
Note on the figures
The phylogenetic trees were constructed using RAxML and drawn using FigTree. The bars indicate the proportion of nucleotide changes between sequences. Reference strains are viruses to which post-infection ferret antisera have been raised. The colours indicate the month of sample collection. Isolates from WHO NICs in ECDC countries are highlighted within boxes. Sequences for some of the viruses from non-EU/EEA countries were recovered from GISAID. We acknowledge all laboratories who submitted sequences directly to the London WHO Collaborating Centre.
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