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Transmission of Fatal H1N1 D225G/N Accelerates Concerns
Recombinomics Commentary 19:05
January 11, 2010

Recently released H1N1 HA sequences have significantly accelerated pandemic concerns.  These sequences have either D225G, D225N, or both, and produce a case fatality rate at or near 100% in many countries.  These receptor binding domain changes are on multiple backgrounds, but the transmission and expansion of these fatal sequences in eastern Europe, including Russia, have increased concerns, as has the "low reactor" status as determined by Mill Hill for a Ukraine sequence.

Some of the earliest sequences with D225G were in the United States last spring and were generally mild.  However, initial cases in the US were usually mild, which may have reflected low viral loads infecting a naïve population.

The concerns regarding D225G and D225N began to increase when sequences were released from fatal cases in Sao Paulo Brazil.  These samples were collected in July, when the flu season in the southern hemisphere was peaking.  Two lung samples were positive for D225G, while a lung and throat sample were positive for D225N.

There already was interest in position 225 for a number of reasons.  D225N  was in seasonal H3N2 and linked to the fixing of adamantine resistance (S31N), while D225G was in 1918 and 1919 samples and linked to a change in receptor binding domain specificities which would target subsets of cells in the lung.  In addition, the polymorphism was jumping from one genetic background to another signaling recombination and increased chances that the polymorphisms would continue to jump to new genetic backgrounds.

Thus, when reports from Ukraine described a high number of fatal cases associated with lung destruction and a hemorrhagic component, involvement of D225G and D225N was predicted.  However, although WHO had sent a team to Ukraine and had sent representative clinical samples to Mill Hill and CDC, WHO regional labs, WHO characterized the sequences changes in Ukraine as insignificant.

However, release of the sequences by Mill Hill confirmed the linkage of D225G in the outbreak in general and in the fatalities in particular.  Sequences from 10 patients were released.  One was from Kyiv and distantly related to the nine sequences from western Ukraine.  Although the Ternopil/Khmelnitsky sequences could be distinguished from the Lviv sequences by a synonymous HA marker, all western Ukraine sequences were closely related.  However, the four fatal cases (two from Lviv and two from Ternopil) had D225G appended onto the common background, which was distinct from the sequences in Brazil and the United States.  Thus, not only was D225G present in all four fatal cases, it was only in the four fatal cases, leading Norway to examine sequences from patients in Norway.  Three with D255G were identified and two had died.  The third was a severe cases who had recovered.  This high level of fatalities led Norway to issue and alert and other countries, like France had similar results (two patients with D225G and both were fatal and one was Tamiflu resistant).

The Mill Hill sequences also suggested that D225N may also be involved in the Ukraine fatalities because one of the fatal sequences in Ternopil, A/Ternopil/11N/2009, had a novel marker that was only found in two other H1N1 sequences at Genbank, and these were the two sequences from New York with D225N.  This associate was confirmed when the CDC released five sequences.  Three were from the same mild cases sequenced by Mill Hill, but the other two were unique and likely from fatal cases. Both had D225N and suggested a relationship between D225G and D225N since all six likely fatal cases in Ukraine had D225G or D225N.

The association of D225G and D225N was more directly supported by sequences from the United States, Mexico, and Sweden, which identified samples with both D225G and D225N.  Thus, once again two different changes were appended onto the same genetic background at a given location, but the background varied from location to location, supporting recombination.  Moreover, in Mexico there were fatal infections with D225G and D225N.  Both were in San Luis Potosi and collected with a day of each other, supporting transmission.

However, although the above data left little dount that the receptor binding domain (RBD) changes were transmitting and jumping from one genetic background to the other via recombination, the ECDC came out with a report at the end of the year stating that the WHO working hypothesis held that each of these changes was independent and due to copy errors within each patient, and the RBD changes did not transmit. 

This position had no support from the data.  The changes at position 225 were only found in 1% of sequences, but were in six of six fatal cases in Ukraine.  Similarly, isolates with both changes were found in two patients at the same location at the same time in Mexico.  The WHO working hypothesis was yet another attempt to explain genetic drift by random mutation, even though the existing data offered no support for such a claim.

The sequences released in the past few days further degrade the WHO working hypothesis as well as the linkage of random mutations to genetic drift.  Mill Hill released sequences from Ukraine and Moldova which also had both changes in the same patients.  In Ukraine the sequences followed the outbreak, which spread to the east.  Samples from the Kyiv area were from lung and each sample had D225G and D225N.  One sample also had D225A.  This clustering of position 225 changes within individual samples is not easily explained by independent and random events.  The same set of two changes was seen in a patient from Moldova, which is adjacent to Ukraine.  In addition, sequences from cases in Russia also had the D225G and D225N changes and were also likely from fatal cases.

Thus, the recent data demonstrates a case fatality rate at or near 100% in multiple countries, with clustering of polymorphisms and patients consistent with transmission and recombination.  Moreover, Mill Hill ran an antigenicity test on one of the Ukraine samples and found it to be a low reactor, raising concerns that changes at position 225 will become more common in the next H1N1 wave, which could have catastrophic consequences, while WHO is trying to construct a defense for its outdated random mutations as an explanation of genetic drift and viral evolution.

The transmission of a deadly H1N1, coupled with a WHO wedded to an outdated paradigm, significantly increases pandemic concerns.

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