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As the 2009 (H1N1) influenza A virus continues evolving, most mutations appear geographically and temporally confined. However, the latest surveillance data suggests emergence of a new prominent mutation, E391K, in the hemagglutinin (HA) that is globally on the rise. Interestingly, when modelled in the context of the available HA crystal structure, this mutation could alter salt bridge patterns and stability in a region of the HA oligomerization interface that is important for membrane fusion and also a known antigenic site. We discuss occurrence of HA-E391K in global surveillance data and associated clinical phenotypes from Singapore ranging from mostly mild to few severe symptoms, including sporadic vaccine failure. More clinical and experimental data are needed to determine if this mutation could alter the biology and fitness of the virus or if its increased occurrence is due to founder effects.
The pandemic 2009 (H1N1) influenza A virus    continues to circulate worldwide, giving rise to concerns about new strains emerging which might cause further outbreaks. As a well-connected international hub, Singapore receives a broad sample of globally circulating influenza strains and 2009 (H1N1) remains the dominant influenza strain. The mutation HA-E391K was first identified in New York in July 2009 and appeared shortly after also in Singaporean samples but only grew rapidly in appearance recently (Figure 1). HA-E391K has been found in samples from 20 countries so far.
New substitutions that are globally found in more than 20% of the sequences with collection date December 2009 in the NCBI Influenza Virus Resource  include HA-D114N, HA-E391K, PA-V14I, PA-K716Q, PB1-K736G, PB1-R563K and PB2-K340N. Analysis of their co-occurrences identifies 9 clusters, 5 of which include HA-E391K (Table 1). All of these variants co-occur with the globally dominant strain (variant (ii) in Table 1) characterized by the 5 substitutions HA-S220T, NA-V106I, NA-N248D, NP-V100I and NS1-I123V. PB2-K340N is also of interest as it frequently co-occurred with the potentially virulent HA-D239G mutation   but this may also be the reason for its increased occurrence due to a sampling bias towards sequencing more severe cases.
Table 1. Co-circulating variants based on frequent marker mutations in December 2009. Residue numberings and mutations are relative to reference strain A/Texas/04/2009(H1N1). Con. … conserved.
Looking at the temporal detail of the variants including HA-E391K, the percentage of co-occurrences with PA-V14I, PA-K716Q and PB1-K736G has changed from September 2009 to December 2009 (56.3%, 68.0%, 57.4% and 34.8% respectively). The HA-E391K co-occurrences with HA-D114N and PB1-R563K on the other hand increased from September 2009 to December 2009 (6.3%, 26.7%, 39.3% to 47.8% respectively). This co-occurrence can be found both in Europe and the USA. The co-occurrences of HA-E391K with PB2-K340N was found only recently in 2 viral strains in December 2009 (one in Poland and the other in Greece). The co-occurrence of HA-E391K with HA-D114N, PB1-R563K and PA-V14I was found in December 2009 in one strain from Spain. Variants with HA-E391K but without the 6 other substitutions were also found in Germany, Nicaragua, Taiwan and the USA.
Phylogenetic analysis of the Singaporean sequences including their closest non-Singaporean international matches shows that sequences with HA-E391K have appeared multiple times in different contexts (Figure 2). While in some cases, the best match is another Singaporean sequence without HA-E391K, in others it is a non-Singaporean sequence already with HA-E391K which could indicate import from or exchange with other global outbreak clusters including HA-E391K. It should be noted, however, that detailed phylogenetic relations are difficult to conclude from this analysis due to the high sequence similarity among the strains. Other sequence clusters that include a second co-occurring mutation among all closely related strains in the respective subtree are found for HA-E391K with HA-D114N (see discussion of global appearance of these coupled mutations above) as well as HA-E391K with V47A which would be indicative of founder effects for these cases. The relative dominance of HA-E391K containing strains in Singapore compared to their average global occurrence in December 2009 (Figure 1) may have originated from founder effects.
The hemagglutinin position 391 corresponds to the HA2-subunit position 47 mentioned by Smeenk and Brown  . In their studies with mouse-adapted H1N1 influenza virus variant A/FM/1/47, the W47G substitution causes a decrease in optimum pH of membrane fusion which led to an increase in necrosis of the bronchiolar epithelium, peribronchiolar lymphocytes, and airway obstruction. This effect can partially be explained mechanistically as the mutation drastically alters the oligomerization interface in a region that undergoes structural changes required for membrane fusion  . As also identified by the authors of another HA crystal structure  published while this analysis was in preparation, the HA-E391K mutation alters this very same region. Additionally we find that the intra-molecular salt bridge of E38 to R336 could be broken to allow an inter-molecular salt bridge instead between E38 from one monomer to K391 in the other (Figure 3). This could weaken stability of the membrane fusion region within an individual monomer while it strengthens the interaction between the monomers. Furthermore, this region was recently identified as highly conserved epitope recognized by antibodies that neutralize the closely related 1918 H1N1 virus by blocking the structural changes associated with membrane fusion . Ultimately, only experimental or detailed clinical data will be able to establish if HA-E391K has the potential to alter the biology of the virus-host interactions and antigenicity or if its increasing occurrence is due to founder effects.
The clinical phenotype for 35 Singaporean samples that had the HA-E391K mutation was available for analysis (Table 2).
*1 relative to reference A/New York/20/2009(H1N1) which additionally differs from A/California/07/2009(H1N1) by the HA mutations P100S, S220T, I338V; *2 low reactor against A/California/07/2009(H1N1)-derived antisera; *3 normal reactor against A/California/07/2009(H1N1)-derived antisera.
Table 2. Summary of HA mutations for patients with at least partial clinical info.
33 of the 35 cases had only mild symptoms, while 2 were severe (intensive care unit or death). Although this would result in 5.7% of cases with the mutation being severe, this number is likely to be heavily overestimated since all severe cases are systematically analyzed in Singapore while mild cases are mainly added through routine surveillance. The age distribution among patients with HA-E391K continues to follow the general pattern of 2009(H1N1) by strongly affecting the younger age groups.
For the severe cases, no additional known mutation, such as HA-D239G (D222G or D225G in alternative numberings  ), was found that could directly explain severity. In both of the severe cases with HA-E391K, we also find a HA-V47A mutation which is structurally located in vicinity (13 Angstrom) of the E391K mutation (Figure 4) and, hence, could contribute to any effects in the region as discussed above. At the same time, there have been three other cases with both HA-E391K and HA-V47A but showing only mild symptoms. As 4 of the 5 cases with HA-E391K and HA-V47A were closely temporally related (within ~2 weeks), the co-occurrence could derive from the same transmission chain/cluster as also indicated by their grouping in the phylogenetic analysis.
Initially, the HA-E391K mutation came to our attention as it occurred in a special case where a patient had been vaccinated but still contracted 2009(H1N1) as evidenced by sequencing. Besides HA-E391K, the sample additionally had the mutation HA-N142D which was previously recognized as antibody escape mutant in the context of H5-type viruses (N142D corresponds to N129D in H3 numbering in Table 2 of reference ). Interestingly, a recent second vaccine failure case showed the same co-occurrence of HA-E391K and HA-N142D. Hence, it is not clear if the vaccine failure in these cases was due to HA-E391K, HA-N142D or patient-specific factors.
For 5 samples of 2009(H1N1) with HA-E391K, reaction towards the vaccine based on strain A/California/07/2009(H1N1) was tested through hemagglutination inhibition assays by the regional WHO collaborating centre in Melbourne. Interestingly, 2 of the 5 samples were reported as low reactor. Although the currently dominant strain is more similar to the early genomic variant A/New York/20/2009(H1N1) than to the common vaccine strain A/California/07/2009(H1N1), these two strains have been previously found to be antigenically closely related by Garten et al. . Consequently, if the common variations between dominant and vaccine strain do not alter antigenicity, the only additional mutation shared among the low reacting samples was HA-E391K. However, it should be noted that 3 of the 5 samples with HA-E391K (one with both HA-E391K and HA-V47A) still showed normal A/California/07/2009(H1N1)-like antigenicity.
Overall, these examples are interesting but not sufficient to significantly link the HA-E391K mutation (with or without HA-V47A) to increased severity or lower vaccine efficacy as this may depend on many other patient-specific factors and the fact that these cases had the mutation could readily be explained by the fact that strains with HA-E391K are currently the most common flavor among all locally available samples.
HA-E391K is a globally fast growing mutation in 2009 (H1N1) samples and could alter the salt bridge pattern and stability in a region of the HA oligomerization interface that is important for membrane fusion and also a known antigenic site. More clinical and experimental data are needed to determine if this mutation could alter the biology and fitness of the virus or if its increased occurrence is due to founder effects.
The authors wish to thank Michael Levitt for constructive discussions regarding the salt bridges involving HA-E391K and the regional WHO collaborating centre in Melbourne for HI testing. Some of the early Singaporean samples downloaded from Genbank were sequenced by the Genome Institute Singapore (GIS) for which we would like to acknowledge Christopher Wong and Martin Hibberd.
NPHL is funded by the Ministry of Health, Singapore and BII by the Agency for Science Technology and Research (A*STAR), Singapore.
The authors declare that no competing interests exist.