Recombination Driving H1N1 D225G and H274Y Spread Recombinomics Commentary 20:08 February 02, 2010
The recently released H1N1 receptor binding domain sequences leave little doubt that homologous recombination of the polymorphisms are producing rapid influenza evolution, which contradicts with WHO working hypothesis that the genetic drift is due to random mutations generated by frequent copy errors. Position 225 has been the focus of attention and dramatic differences between high D225E levels in western Europe and high D225G/N in eastern Europe highlights these differences, which is increased by the requirement for multiple introductions due to sub-clade differences. However, a review of homologous recombination in influenza will provide additional information on these striking differences.
The random mutation model was seriously discounted by the emergence of Tamiflu resistance (H274Y) in H1N1 seasonal flu in the 2007/2008 season and the fixing of H274Y in the 2008/2009 season. The random mutation model was based in part by lab experiments on drug resistance which identified import changes linked to resistance by selecting organism that emerged from drug treatment, which put heavy selection pressure on genomes that did not have resistance mutations. This experimental model was validated in Japan when children were treated with sub-optimal doses of Tamiflu. Resistance developed at multiple positions in the targeted NA gene, but the resistance was largely limited to the H3N2 in patients who were treated with the sub-optimal dose of Tamiflu. Use of the correct dose of Tamiflu quickly eliminated the resistance and Tamiflu resistance in H3N2 is now extremely rare.
In early 2008, Norway reported a high level (over 60%) of Tamiflu resistance (H274Y) in patients infected with H1N1. Unlike the results in Japan, the patients had not been treated with Tamiflu, and Tamiflu was rarely used in Norway for seasonal flu. Moreover, all of the resistance was in H1N1 and all was due to the C to T change in the first position of the 274 codon (275 in N1 numbering). This initial report was followed by reports of widespread resistance which was at highest frequencies in northern Europe, where Tamiflu usage is low, and at 3% in Japan, where Tamiflu use is higher.
Moreover, all of the resistance was in H1N1 and involved the same genetic change. Detailed analysis showed that the H274Y was present as early as 2006, but at a lower level. However, although the earlier cases involved different sub-clades, all were H1N1 and most were in patients who had not been treated with Tamiflu. The high levels in 2008 were largely due to one Brisbane/59 sub-clade, but there were many examples in other Brisbane/59 sub-clades signaling multiple introductions. Moreover, the evolving sub-clades were acquiring polymorphisms present in the Hong Kong strain, which was co-circulating, especially in Asia. The jumping of these polymorphism from one sub-clade to another signal recombination, which was strongly support by acquisitions of adjacent polymorphisms, including synonymous polymorphism which would not be under strong selection pressure.
The fixing of H274Y involved the acquisition of a receptor binding domain change A193T, which first appeared on the Brisbane/59 strain in the 2007/2008 season, but then appeared on the dominant sub-clade in the southern hemisphere. This sub-clade subsequently was dominant in the 2008/2009 season, which led to the fixing of H274Y in H1N1. All isolates had A193T on HA and H274Y on NA, and sub-clades were created by HA changes flanking A193T (at positions 187, 189, and 196). The association of anti-viral resistance with receptor binding domain changes had also been seen in H3N2, which adamantine resistance (M2 S31N) was linked to another change at HA position 193 (S193F) as well as D225N.
When H1N1 pandemic flu emerged, there was concern that H274Y o seasonal H1N1 would be acquired by the pandemic strain. A recombination mechanism predicted that H274Y would be acquired and it would then spread to multiple sub-clades and eventually pair up with a receptor binding domain change. Others expected resistance to be acquired by reassortment, where the entire swine N1 gene would be swapped for a human N1 gene with H274Y. However, to date there are no examples of pandemic H1N1 acquiring a human N1 or any other human flu gene. The current constellation of 5 swine, 2 avian (PA and PB2) and 1 human (PB1) remains unchanged. However, H274Y has been acquired, has jumped from one sub-clade to another, and has paired up with receptor binding domain changes at position 225 (D225E and D225G).
In addition to the spread and fixing of H274Y, additional concerns have been raised over the emergence and spread of receptor binding domain changes at position 225. The first example of Tamiflu resistance in a patient not treated with Tamiflu was in a traveler from San Francisco to Hong Kong. Although she had not taken Tamiflu, the pandemic H1N1 had H274Y. Moreover, it also had D225E, raising concerns of a repeat of the evolutionary events in seasonal H1N1. Initial searches for other examples were negative, but the frequent acquisition of D225E was found, especially in travelers from the United States. However, examples of additional isolates with H274Y and D225E have been identified in Tennessee and Shiga, Japan indicating this combination is transmitting.
Similarly the high levels of D225E in western Europe also indicate it is transmitting and the presence on multiple sub-clades indicates signals independent introductions, which is most easily facilitated by recombination.
However, the jumping to sub-clades which contain isolate with wild type receptor binding domains is most pronounced for D225G/N isolates. Sequences from a large number in Ukraine and Russia have now been released. These isolates are almost exclusively from fatal cases and cluster in time, space, and phylogeny, signaling transmission and recombination, which is inconsistent will recent remarks by WHO, maintain that D225G was spontaneous sporadic and due to copy errors, which simply is not supported by the published sequences.
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