Qinghai H5N1 Polymorphism Acquistions Via Recombination
Recombinomics Commentary 20:10
March 24, 2008
The recent recognition of the sudden appearance of the Tamiflu resistance marker H274Y on multiple genetic backgrounds in H1N1 seasonal flu has raised issues regarding the validity of one of the basic tenets of influenza genetics, which maintains that genetic drift is due to selection of random mutations generated by copy errors. The Tamiflu resistance has appeared in areas not known for Tamiflu treatment of seasonal flu, is limited to position H274Y in H1N1, and has become widespread in certain regions.
However, this type of sudden appearance of influenza polymorphisms has been noted previously. One of the most dramatic examples is the appearance of another neuraminidase polymorphism, on multiple H5N1 genetic backgrounds. This change, G743A is silent. Thus, the lack of a change in the NA protein sequences removes escape from vaccines or immunological response directed against the NA protein as a selection mechanism for the change.
In 2006 G743A was a regional marker for clade 2.2 isolates in southern Germany, Switzerland, and France. It was present in the dozens of the closely related NA sequences, from the three countries. However, the distribution was quite localized. Other subclades were reported in Germany. One was in northern Germany and closely related to isolates in Denmark, Sweden in Scotland. Another was in southern Germany and was closely related to isolates in Italy, Slovenia, and the Ukraine. However like all other clade 2.2 2006 isolates in Europe, the Middle East and Africa, the isolates did not have G743A, other than one isolate in western Africa.
At the end of 2006, the Gharbiya cluster was reported in the Nile Delta in Egypt. The three family members were fatally infected with H5N1 and the NA sequences from isolates from two of the family members had the Tamiflu resistance marker, S294N. This discovery led to more widespread surveillance in Egypt, which included more NA sequencing. Although additional examples of Tamiflu resistance were not found, three isolates from chickens in Gharbiya were G743A positive. These isolates were collected in mid-February and represented the first examples of G743A. However, although all three birds were from Gharbiya, the isolates represented two distinct sequences. One sequence was closely related to the Ghribiya cluster. The initial isolate was a mixture, which was confirmed by sequencing of plaque purified clones. The minor species matched the other two chicken isolates from Gharbiya, while the major species differed from the Gharbiya cluster consensus sequence at two positions. One of the two differences was G743A. The other chicken sequence in Gharbiya was similar to isolates collected in mid-2006 from the adjacent governorate, Monofiya. The 2007 isolates differed from the 2006 isolates at three positions, and one was G743A. There two sets of 2007 sequences however were distinct and had 11 differences. Thus, although the 2007 isolates were from the same location at the same time, the sequences were different, yet had acquired G743A, even though only 2 or 3 changes were present in each series.
Explaining the same change on two distinct genetic backgrounds because of random mutations would be difficult, especially since the change was silent. However, the explanation would be more difficult because of the same changes at additional locations at the same time was repeated in Kuwait and Russia.
In mid February of 2007 the Kuwait and Moscow outbreaks involved very distinct clade 2.2.3 sequences. In Kuwait, the sequences traced back to a massive wild bird outbreak at Uvs Lake in Mongolia in the summer of 2006. These sequences did not have G743A nor did related sequences from an outbreak in wild birds and poultry in south Korea at the end of 2006. However, all isolates from Kuwait did have G743A. The consensus sequence in Kuwait differed from the Uvs Lake sequences (from wild birds in Mongolia and Tyva) at six positions, and one of the six was G743A.
The outbreak in Moscow was also clade 2.2.3, but the sequences did not trace back to the summer outbreak in Mongolia, but instead traced back to the H5N1 outbreak a year earlier in Azerbaijan. The number of differences between the 2006 isolates in Azerbaijan and the 2007 isolate in Moscow was also six, and one of the six was G743A (a partial NA sequence from Moscow was also released, and it also had G743A).
Thus, in February 2007 there were outbreaks in Egypt, Kuwait, and Russia and in each series one of a small number of changes was G743A. Moreover, in Egypt the addition of G743A was on two distinct backgrounds, which also differed from the two distinct clade 2.2.3 isolates.
However, shortly thereafter G743A appeared on additional Egyptian backgrounds. Another branch on an NA phylogenetic tree hand been formed by isolates further to the south which had a 3 nucleotide deletion among multiple changes that distinguished this group from other Egyptian isolates, In April, two siblings were infected with H5N1 which had acquired G743A. These sequences differed from the consensus sequence of the earlier isolates at two positions, and one of the two was G743A. At the same time there was an H5N1 outbreak in Ghana. Although these isolates were the first reported from Ghana, they were related to earlier isolates from the Ivory Coast, which were isolated at the end of 2006 and did not have G743A. The Ghana NA sequences differed at six positions, and one of the six positions was G743A.
The G753A acquisition was subsequently found in another genetic background in Nigeria, as well as all published NA sequences from Uvs Lake related sequences from three locations in Germany, the Czech Republic, Krasnodar in Russia, and Saudi Arabia. Thus, G743A became widespread in 2007 via acquisition of this polymorphisms onto multiple clade 2.2 genetic backgrounds, which cannot be easily explained by random mutations, especially since the number of changes in each series was six or less.
Like the appending of H274Y onto multiple H1N1 backgrounds in seasonal flu in 2007 and 2008, the addition of G743A onto multiple H5N1 genetic backgrounds and be easily explained by recombination. These changes raise serious questions about the basic tenet of influenza genetics that explains genetic drift through selection of random mutations.
As more clade 2.2 sequences are released, additional examples, including recent 2007 and 2008 sequences from Egypt, leave little doubt that recombination is the driving force behind genetic drift in influenza and other rapidly evolving genomes.
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