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Qinghai H5N1 Evolution Revolution

Recombinomics Commentary
May 10, 2007

The recent H5N1 sequence data out of the Middle East and Western Africa further support a paradigm shift in the understanding of evolution.  The recent data strongly indicate that single nucleotide changes, previously thought to be due to random mutations, are really genetic changes acquired by recombination and are not due to de novo mutations resulting from copy errors.  The selection of copy errors represents the previous understanding of genetic evolution and drug resistance, but the current data indicates that rapid change is through selection of recombinants.

H5N1 evolution provides a model for rapid evolution in a natural setting.  The growing sequence database provides examples of how influenza genes evolve over time, and the recently discovered Qinghai strain (clade 2.2) firmly ties this evolution to migratory birds.

Sequence data provide the initial evidence against the role of de novo random mutations in influenza evolution.  Genetic drift allows the virus to escape immune responses. H5N1 is  the fastest evolving influenza serotype and has created the greatest concern because of the associated high case fatality rate in infected hosts, which is coupled with rapid change into an expanding geographical reach and a growing host range.  Recent sequence data identified clear cut recombination in H5N1 isolates in China, and recent swine sequences identified sequences copied with absolute fidelity for over 25 years.  These two observations raised serious questions about the role of random mutations in the rapid changes seen in both seasonal and pandemic influenza genes.

Similarly, polymorphism tracing identified the rapid movement of polymorphism from one genetic background to another, which followed identifiable pathways that coincided with the movement of migratory birds.

The role of migratory birds became increasingly clear after the H5N1 Qinghai outbreak, almost exactly two years ago.  On May 9, 2005 178 dead bar-headed geese were reported at Qinghai Lake in central China.  Although H5N1 involvement was initially denied, a novel strain was identified in five species of long range migratory birds.  Eventually the number of dead birds at Qinghai Lake exceeded 5000.  Most were bar headed geese that could travel 1000 miles in 24 hours.  The establishment of H5N1 in such long range migratory birds set the stage for a rapid expansion of the H5N1 global reach.

A few months later, H5N1 was reported for the first time in Russia, Kazakhstan, and Mongolia.  When sequence data indicated that all three outbreaks were due to the Qinghai strain of H5N1 in long range migratory birds, it was clear that a dramatic increase in the H5N1 reach had begun.

The regions in Siberia and Mongolia were linked to migratory pathways that would move H5N1 into Europe, the Middle East, and Africa.  Within 12 months H5N1 had been reported for the first time in almost 50 countries west of China and all outbreaks involved the Qinghai strain.

This rapid spread created an experiment of nature for the study of influenza evolution in a natural environment.  H5N1 had not been reported in any of these countries previously, and the newly reported sequences included newly acquired polymorphisms that were regionally specific.  The polymorphisms could be traced to determine the origins of the changes to confirm the role of recombination in the acquisitions.  As expected, the vast majority of the newly acquired polymorphisms were already present in the sequence database.  Most could be found in other H5N1 isolates, although contributions from low path polymorphism were also seen.

However, the greatest insight came from closely monitoring changes in a large number of isolates from the same region over a limited time frame.  This opportunity was presented by the sequences generated by US NAMRU-3 in Egypt.  Like most of the countries in Europe, the Middle East, and Africa, Egypt reported its first case of H5N1 in poultry in February, 2006.  However subsequent analysis of an isolate from a healthy teal indicated that Qinghai H5N1 was already in the Nile Delta in December, 2005.  Egypt also lies under overlapping migratory bird flyways, allowing for multiple introductions and significant genetic mixing via recombination.

After the outbreaks in the winter / spring of 2006, detection of H5N1 decreased.  New infections in poultry and humans were reported in the fall of 2006 signaling a new season and a new set of H5N1 sequences.  The new sequences were more genetically complex and the newly acquired polymorphisms were frequently found in earlier Qinghai isolates, although the regional markers seen in early 2006 were also present in 2007.  The recently released sequences from 2006 H5N1 isolates in Israel and Gaza indicated that the regional markers seen in Egypt extended to Israel and Gaza, as had been seen earlier in a human isolate from Djibouti.  The 2006 isolates from Egypt, Djibouti, Israel, and Gaza formed a genetic baseline for H5N1 polymorphisms in the region, so newly acquired polymorphisms in the 2006/2007 season were easily identified.  The large number of poultry and human samples collected by NAMRU-3 provided a real time view of the H5N1 evolution.

The newly acquired sequences were readily found in H5N1 isolates in eastern Asia, including changes in the receptor binding domain as well as oseltamivir (Tamiflu) resistance, providing further evidence for acquisitions via recombination.  The donor sequences in eastern Asia were on H5N1 isolates, but these isolates were genetically distinct from the recent isolates in Egypt, which had the regional markers from the prior season.

Similarly, as more sequences from Qinghai isolates from other countries were released, it became increasingly easy to find the newly acquired polymorphism in Egypt in other locations, but the polymorphism was being acquired individually. One of the changes in Egypt was M230I which was encoded two different ways. One matched H5N1 in Asia, while the other matched H7N3 in Europe.  However, recent sequences showed that the Asia version of M230I was in a Qinghai isolate in a German eagle owl, indicating both versions that appeared in the Nile Delta in Egypt in the 2006/2007 season were in wild birds in northern Europe in the 2005/2006 season.

The sequences in Egypt began to form new branches on the phylogenetic tree and the branches increased in size.  One branch was created by sequences from the Gharbiya cluster which involved three fatally infected family members.  These sequences had two changes in or near the receptor binding domain, and these polymorphisms were matched by an isolate collected on February 15, 2007.  However, that isolate had another change that was not seen in the Gharbiya cluster.  That change, NA G743A was also seen in the eagle owl with M230I, but it was in a number of German isolates which had clear regional markers that were not present in the Egyptian sequences. Moreover, the NA G743A was also in two additional isolates from Gharbiya collected on February 15,2007, but the two additional isolates were clearly distinct from the Gharbiya cluster.

The same polymorphism subsequently appeared in three human isolates from central Egypt.  These isolates formed two additional branches and were easily distinguished from the branches linked to Gharbiya.  Moreover, two of the isolates from central Egypt were from siblings and mapped to a branch of isolates with a 3 BP deletion in HA.  This deletion was an exact match to 2006 isolates from Hunan province in China which were markedly different from the sequences in Egypt.  The siblings with G743A also had the 3 BP deletion in HA, providing more compelling data for acquisitions of individual polymorphisms via recombination.

The presence of G743A in six isolates on four branches of the HA or NA phylogenetic trees provide compelling evidence for evolution by acquisition of single nucleotide changes by recombination.  This mechanism was further supported by the acquisition of G743A by two Qinghai isolates in Russia (Moscow) as well as three Qinghai isolates in Ghana.  As was seen in Egypt, closely related sequences collected earlier did not have G743A, providing more evidence for the recent acquisition of G743A onto six genetically distinct Qinghai backgrounds.

The evolution by acquisitions of single nucleotide changes via recombination represents a paradigm shift creating an evolution revolution.

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