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H1N1 Greek and Russian D225G Sequences Identical
The 11 Mar. 2010 at 07:30Last newsComments (0)Add a comment
Recombinomics Commentary 18:50
March 10, 2010
Recent sequences from the National Influenza Centre for northern Greece include 9 sequences from 2010, of which 3 had D225G. Three more November isolates had D225G, including A/Thessaloniki/2812/2009. Three of the isolates had D225G as the only recent change, but the other three has polymorphisms found in three different sub-clades. The above sequence was an exact match (see list here here here here) with A/Bryansk/IIV2971/2009, which had been collected 11 days earlier from a lung sample, suggesting the Russian sequence was from a fatal case. Clinical information from the Greek isolate was not included, but the samples were from a study of severe and fatal cases in Greece, suggesting the D225G positive samples were from such cases.
The identity between the samples collected in Russia and Greece 11 days apart provides compelling evidence that the H1N1 is transmitting with D225G and is not due to spontaneous mutations, which is the WHO working hypothesis. Transmission is also supported by sequences from autopsy lung in Ukraine, where 27/37 samples had D225G, D225N, or both. The same association of these changes with fatal cases was seen in the Duke death cluster where 3 of 4 samples from the first three patients, who were on the same ward, had D225G or D225N.
The three D225G cases in 2010 is the largest number reported to date from a single country this year and the nine cases from 2010 is also the largest number of sequences from a given country. D225G has been reported in a 2010 Russian isolate, but thus far there have been few 2010 sequences made public.
However, the finding that D225G is in a Ukraine low reactor raises concerns that the frequency of D225G in 2010 cases will be markedly higher than 2009.G158E H1N1 Low Reactor In South Korea
The 10 Mar. 2010 at 09:39Last newsComments (0)Add a comment
Recombinomics has the storyRecombinomics Commentary 23:54
March 9, 2010
Recently released NIID sequences at GISAID include A/GYEONGGI/4478/2009, which has G158E and was collected on 9/11. When G158E was reported in sequences from Germany, the CDC and Mill Hill independently determined that the isolates were low reactors, which was linked to G158E since it was the only non-synonymous HA change. NIID also released a series of more recently collected isolates in Japan which also had G158E, which dramatically increased the number of public sequences with G158E. Included in the isolates from Japan were sequences with D225N or H274Y. These low reactors with clinically significant markers were in addition to isolates with the combination of G158E and D225G found in Italy and Russia.
The jump in these low reactor markers in Asia adds to the jump reported in the United States. In the latest CDC report, the number of low reactors increased from 2 to 5. An increase in low reactors was expected, as more of the target population develops immunity against wild type H1N1. However, the low reactor, receptor binding domain, and anti-viral markers have been actively jumping from one genetic background to another via recombination, which increases the number of different combinations on different genetic backgrounds.
This rapid evolution raises concerns that there will be widespread vaccine failure against isolates that have resistance to anti-virals and cause more severe and fatal infections.H1N1 Low Reactor G158E D225N Recombinant In Japan
The 09 Mar. 2010 at 09:40Last newsComments (0)Add a comment
Recombinomics Commentary 07:44
March 9, 2010
The recently released sequence, A/Yamagata/721/2009, represents a new recombinant with the low reactor polymorphism G158E and D225N. This sequence follows release of recombinant sequences with G158E and D225G in Russia and Italy. A Ukraine sequence with D225G has also been designated a low reactor by Mill Hill, which when recombined with G158E raises concerns of serious vaccine failure.
D225G has been found to be strongly associated with severe and fatal cases in Norway as well as Ukraine. Egg isolates from milder cases have also been found to contain D225G, which was not seen in direct sequencing of the clinical samples or isolates grown in mammalian cells. However, D225G, has affinity for gal 2,3 receptors found in chicken embryos in eggs, so the egg isolates may represent selection of isolates with D225G and may be a more sensitive assay for samples with low levels of D225G. recent statements by the CDC on the number of patients testing positive for D225G do not include patients with D225G detection limited to egg isolates, indicating the CDC is using the negative data from assays that select against D225G, to nullify detection of D225G in eggs which selective for the change. The use of a negative result from a less sensitive assay to trump positive results from more sensitive assays raises serious concerns on the generation and interpretation of sequence data. This concern is increased when the above “logic” is applied to an important marker like D225G.
Thus, the D225G may be circulating widely but silently because of the minimal use of eggs to expand H1N1 isolates. D225G appears to be on the rise, and this increase may accelerate because of the possibility that D225G confers low reactor status, and D225G recombination which adds another low reactor polymorphism, G158E.
These recent results raise serious question about H1N1 surveillance represented by public sequences, and continue to be hazardous to the world’s health.CDC Reliance on Negative D225G/N Data Raises H1N1 Concerns
The 07 Mar. 2010 at 23:11Last newsComments (0)Add a comment
Recombinomics Commentary 19:32
March 7, 2010
Some countries have reported the D222G mutation only in mild cases, while others have seen it as statistically linked with severe illness, but the latter didn't rule out possible confounders, Cox said. She reported that the CDC has found the mutation in a total of eight cases, of which five were nonfatal. Some of the nonfatal cases were mild.
The above CDC comments, in response to the Norway study showing strong linkage between D225G and severe (3) and fatal (11) cases compared to mild (0) cases, mentions a subset of CDC sequences from US cases. The acknowledgment of only 8 cases of D225G in sequences generated by the CDC, when at least 15 D225G positive US sequences have published indicates the CDC is relying on negative data from direct sequencing to exclude positive sequences from isolates grown in eggs. Reliance on negative data is dangerous when the negative data is based on an assay that lacks sensitivity, and is refuted by positive data from virus grown in eggs as well as mammalian cells.
Others have identified D225G in direct sequencing. The most challenging was the generation of sequences from samples collected from fatalities in the 1918/1919 pandemic. Since the virus was degraded and fragmented, it was not possible to grow the virus. Instead the samples were sequenced directly and five HA sequences were generated, all of which covered the receptor binding domain. Two sequences were from London (1918 and 1919) while 1918 sequences were generated from New York, South Carolina, and Alaska. This direct sequencing approach identified D225G in two of the five sequences, including the only sequence from 1919. The D225G in 1918 New York sequence demonstrated that D225G was widespread and present in both waves tested. Since H1N1 in 1918/1919 was readily transmitted and resulted in an estimated 50 million casualties, the finding of D225G in 40% of the 1918/1919 samples tested is significant.
However, the presence of D225G and or D225N in direct sequences from autopsy lung samples in Ukraine is also striking. 30 of 37 samples had D225G, D225N, or both. Moreover, like Norway, virtually all samples positive for D225G or D225N were from fatal cases. Thus, the significant association of D225G with fatal cases has been demonstrated by direct sequencing, as well as from sequences from virus grown in mammalian cells, as was done in the study from Norway.
However, the CDC has also generated D225G and D225N sequences in mammalian cells even though the direct sequencing was negative. This dataset was generated in the fatal cluster at Duke Medical Center. Four patients were infected the Tamiflu resistance H1N1 that also had a rare HA marker, Y233H. Thus, it was clear that all four patients were infected by the same H1N1. The direct sequencing produced partial HA sequences with a wild type receptor binding domain. Three of the four patients died and three of the four also had samples collected prior to Tamiflu treatment, which were likely the samples collected from the first three patients which were collected within 1 day of each other. Four samples were collected from these three patients and three of the four samples had D225G or D225N casting serious doubt on the true lack of D225G or D225N in the original samples as indicated in the sequences released by the CDC. Moreover, both samples collected from the same patient had increasing levels of D225G. The first collection had D225G as a mixture with wild type, while the second collection had D225G in the absence of D225. The sequence from the original sample from this second collection was not released.
The detection of D225G and D225N in isolates from mammalian samples raises serious concerns about the reliance on negative data from direct sequences, as was seen in the Duke samples, as well as April samples which were positive for D225G in virus grown in eggs.
The assumptions that the D225G and D225N in lab cultures are artifacts have serious consequences for the selection of vaccine targets as well as the monitoring of clinically relevant polymorphisms, such as D225G and D225N.
Independent evaluation of the direct sequence from the second collection from the Duke patient, as well as the tracings of the underlying data for all sequences from original samples which were positive for D225G/N in viral cultures would be useful. The reliance on negative data for public comments and policy continues to be hazardous to the world’s health.