The Golden Thread

Recombinomics Commentary
Page 2 of 3

Author:  Siam [ Sat Nov 21, 2009 12:26 pm ]
Post subject:  Re: Recombinomics Commentary

Siam: Ah Dr Niman you have ruined my weekend.

1918 RBD D225G in Lung Cases in Ukraine and Norway
Recombinomics Commentary 11:29
November 21, 2009

For the two 1918 HA variants, the South Carolina (SC) HA (with Asp190, Asp225) bound exclusively alpha2-6 receptors, while the New York (NY) variant, which differed only by one residue (Gly225), had mixed alpha2-6/alpha2-3 specificity, especially for sulfated oligosaccharides.

The above description is from a paper analyzing receptor binding domain differences in sequences from the 1918 pandemic. The New York variant had D225G, the same change found in lung tissues from fatal swine H1N1 sequences in Brazil, Ukraine, and Norway. The above result clearly demonstrated a change in receptor specificity for D225G, which was present in A/New York/1/1918 and A/London/1/1919, demonstrating the same change I 1918 that has been described in 2009. Although WHO stated that this change was "not significant" in the Ukraine samples, it was associated with the fatal cases and is cause for concern. The concern was increased by the announcement from Norway indicating the same change was found in fatal H1N1 lung infections there also.

Although there have been comments that this change was "spontaneous" and did not spread, the finding of the same change in all four deceased patients in Ukraine from two distinct locations, indicates it did spread, as did the finding of the same change in multiple cases in Brazil and Norway. Although the concept of "random mutation" has been used to explain away the sudden appearance of the same polymorphism on multiple backgrounds, the appearance via recombination is a much stronger argument for the same change to appear at multiple locations at the same time.

The spontaneous mutation theory, which is the foundation of WHO policy and statements on significance of changes relies heavily on a "selection" component, arguing that the same change keeps appearing on different backgrounds because of string selection pressure. However, this same phenomenon was described for a silent mutation on H5N1, which offers no clear selection pressure. Similarly, a silent change was also found in seasonal H1N1 in sequences that had acquired the Tamiflu resistance marker, H274Y. Thus, these silent (synonymous) changes string argue against a coincidental spontaneous mutation, and instead argue that this acquisition is concurrently acquired because of a widespread common donor.

The concept of acquisition via recombination has serious implications for the current pandemic. It was used to predict the D225G change, in part because the change was "in play" and appearing in July/August sequences at increasing frequency, even though the H1N1 sequences represented different genetic backgrounds. Similarly the clusters of Tamiflu resistance in Wales and North Carolina are also driven by recombination, as happened when the identical change was acquired in H1N1 seasonal flu in patients who were not taking Tamiflu (oseltamivir).

Thus, the concept of recombination predicts that the D225G receptor binding domain change, and the H274Y Tamiflu resistance change, which continue to spread via recombination

Author:  Siam [ Tue Nov 24, 2009 6:41 pm ]
Post subject:  Re: Recombinomics Commentary


Spontaneous Mutation Media Myth
Recombinomics Commentary 07:39
November 24, 2009

The mutations appear to occur sporadically and spontaneously. To date, no links between the small number of patients infected with the mutated virus have been found and the mutation does not appear to spread.
The above comment from the WHO briefing on D225G (aka D222G) in Norway describes how the "mutations appear". However, this appearance is based on an outdated view of influenza evolution, which maintains that all newly acquired drift "mutations" are based on copy errors. For D225G, this would require the same copy error to occur again and again on multiple backgrounds, which simply is not reality based.

Although the "random mutation" explanation is one of the basic tenets of the WHO and CDC view of influenza evolution, this explanation is only viable in the absence of data. Extensive influenza sequence data moved this hypothesis into the indefensible category years ago, but it remains at the core of WHO explanations of drift variants, such as the comments above.

The "random mutation" and failure to spread would require each detection to be an independent event. Thus, in Norway, the same copy error would have been made in each of the three patients with D225G. Similarly, the same error would be required for each of the four fatal cases in Ukraine. Moreover, the same error would be made in the vaccine target, A/California/7/2009, because one of the 2:6 reassortants also had D225G. As the number of sequences with D225G increases, the likelihood that the same error happens again and again, among a very small number of differences (for HA in Ukraine, the only non-synonymous change was D225G), becomes untenable.,

The appearance of D225G on multiple genetic backgrounds of H1N1 parallels the sudden appearance of H274Y on multiple backgrounds of pandemic H1N1, which follows the same scenario of H274Y on multiple seasonal flu backgrounds in patients not taking Tamiflu. The acquisition of H274Y was readily explained by recombination, which led to the acquisition of key changes that were on clade 2C and then jumped to a clade 2B background. In addition to the H274Y jump to seasonal flu in patients not taking Tamiflu, silent changes were also observed, which also discounted heavy selection pressure favoring these changes.

For D225G, the change was present in one of the earliest isolates in the United States. It could jump from one background to another via recombination between sequences that are closely related. As a result, the new acquisitions lead to a new single nucleotide polymorphism, which looked like a point mutation, but was really recombination between closely related sequences.

Thus, both H274Y and D225G move from one genetic background to another via recombination. A new spontaneous mutation is not required for each isolate and the same sequence in a given area is just due to clonal expansion of an isolate with the new acquisition. This could be seen in the sequences from Ukraine. The Ternopil isolates had a marker found in all Ternopil isolates, including those from nasal washes that did not have D225G. The receptor binding domain change was appended onto this background. The same change was also on Lviv sequences from fatal cases which did not have the Ternopil marker. Thus, D225G moved from a Lviv to a Ternopil background via recombination (or vice verse). Moreover, the frequent jumping of the same polymorphism from one background to another allowed fro the prediction that D225G would be found on the Ukraine sequences. This type of concurrent acquisition has also been described for a silent mutation in H5N1, which again would be widespread in the absence of an obvious clear selection pressure.

Thus, the movement of the same polymorphism via recombination is common. It explains the sudden appearance of the same marker on multiple genetic backgrounds, and forms a basis for predicting changes.

However, the reliance on a "random mutation" produces "surprise after surprise" among influenza "experts" and creates "appearances" such as spontaneous mutations and lack of transmission which are not based on reality.

Author:  Siam [ Tue Nov 24, 2009 6:42 pm ]
Post subject:  Re: Recombinomics Commentary


H1N1 RBD Changes D225G and D225E in Norway

Recombinomics Commentary 17:39
November 24, 2009

Twenty-five HA sequences from Norway have been released at Genbank. The isolates were collected between May and October and include six sequences with receptor binding domain changes. All changes are at position 225, but 5 of the 6 have D225E, while the sixth has D225G as well as wild type D225. Outcomes are not included in the characterization sheet, but it is not likely that the two fatal and one serious cases described in media releases last week were samples with D225E because the WHO briefing on Norway presented a country list that matched D225G reports.

D225E is not as well characterized as D225G, which was in 1918/1919 isolates and was characterized in receptor binding studies which demonstrated that D225G targeted alpha 2,3 receptors, like those in lung, as well as alpha 2,6 receptors. D225G was also found in four of four fatal cases in Ukraine.

The finding of D225G in Norway (A/Norway/2924/2009) as a mixture with wild type sequences confirms that isolates with receptor binding domain changes can be circulating as mixtures and the sequence identified will be dependent on the tissue sampled and when it is sampled. Thus, Infections with a high frequency of D225G will likely be quickly cleared from the upper respiratory tract because viruses with D225G will quickly go to the lungs, which wild type sequences will be more quickly cleared from the upper respiratory tract. Thus a nasopharyngeal swab may be negative if all not cleared virus has moved to the lungs, or be positive for the wild type if depleted wild type H1N1 is remaining at the time of swabbing. Thus, the true level of D225G may be grossly under estimated by nasopharyngeal swabs, and the one sample positive for D225G may reflect the rare instances where D225G could still be detected in a nasopharyngeal swab.

These recent sequences provide additional data for widespread isolates with receptor binding domain changes at position 225, but the true distribution may require sampling more representative of virus that has infected the lower respiratory tract, including lung.

gb|CY052015.1 A/Norway/4023/2009
gb|CY052007.1 A/Norway/3478/2009
gb|CY051991.1 A/Norway/3059/2009
gb|CY051987.1 A/Norway/2690/2009
gb|CY051986.1 A/Norway/2674/2009

Author:  Siam [ Tue Nov 24, 2009 6:44 pm ]
Post subject:  Re: Recombinomics Commentary


Ukraine Dead Approach 400 - D225G Spreads
Recombinomics Commentary 23:30
November 24, 2009

1,679,237 Influenza/ARI

99,661 Hospitalized

397 Dead

The above figures from the latest daily update from the Ukraine Ministry of Health support a decline in the rate of increases of cases and deaths, but the total is now almost 400 fatalities (see map). The spread was likely slowed by the country-wide closing of schools along with warmer weather. However, it is likely that the virus will return as temperatures drop and the traditional flu season begins, although it is unclear if seasonal flu will be in circulation in 2010.

The receptor binding domain change, D225G, was in four of four sequence from fatal cases, raising concerns that the 2,3 alpha specificity of D225G drove the H1N1 to the lungs and the total destruction. Three patients in Norway were said to also have D225G, and two of the three died while the third had been in serious condition. 25 HA sequences from Norway were deposited at Genbank, but only one had D225G, and it was a mixture. In Brazil both patients with D225G in lung samples had died, and the case in China had been in serious condition.

However, the severity of the infection may be related to the ratio of sequences with and without D255G, as well as viral load, because milder cases involving D225G have also been reported in the United States and Hong Kong.

More detail on additional cases, including sequences from upper and lower respiratory tract from the same patient would be useful.

Author:  rutsuyasun [ Tue Nov 24, 2009 7:00 pm ]
Post subject:  Re: Recombinomics Commentary

Ukraine Dead Approach 400 - D225G Spreads
Recombinomics Commentary 23:30
November 24, 2009

1,679,237 Influenza/ARI

99,661 Hospitalized

397 Dead

CFR = .02%

Author:  Siam [ Fri Nov 27, 2009 3:40 pm ]
Post subject:  Re: Recombinomics Commentary


H1N1 Re-infections Raise Pandemic Concerns
Recombinomics Commentary 02:30
November 25, 2009

Dr. Debra Parsons, a pediatrician at Kid Care West in Cross Lanes, was met with reactions of doubt from local health officials last month when she said two flu tests had come back positive for H1N1, or swine flu.

Parsons first came down with the virus, complete with all the telltale symptoms, in August.

Her son became ill at the same time with the same symptoms. Figuring they had the same bug, Parsons tested herself to see what it was.

The test came back positive for Influenza A, so the lab at Charleston Area Medical Center sent it to be sub-typed. Parsons was positive for H1N1.

Parsons and her son recovered, but in October they started having the same symptoms, but they became much worse.

They were both tested this time, and the results were the same -- they were positive for Influenza A and then H1N1.

"It was swine flu both times," Parsons said.

The above comments on lab confirmed re-infection of two family members two months apart by swine H1N1. These confirmations are supported by many anecdotal reports of similar re-infections. The timing of these infections allowed for easy identification, because at the time there was no seasonal flu, so identification of infections was straight-forward. Moreover, such infections in school aged children and parents are common because H1N1 infections exploded when school began.

At the time however, the H1N1 virus was evolving slowly, reflecting an ease of infection of a naïve population. The jump from swine H1N1 into humans allows for infections with low doses of virus. Low concentration of virus produces a mild infection and a weak antibody response. The rapid spread creates widespread antibody, but the low level allows for re-infection from individuals with a higher viral load. A higher viral load can be created in a school environment, where some students could be infected multiple times because of frequent contact with infected students.

Thus, the increased viral load could overcome the weak immune-response and re-infect those infected earlier, leading to a second wave. However, the higher viral load leads to more serious infections, especially for these not infected in the first wave. Consequently more previously health young adults develop more serious symptoms, leading to an increase in hospitalizations and deaths. The higher viral load, especially when combined with receptor binding domain changes such as D225G can lead to the type of cases seen in Ukraine, where a high percentage of young adults develop infections that destroy both lungs in a matter of a few days.

In many areas, including Ukraine, this wave is subsiding, but the holiday season will lead to new infections by viruses with regional markers, leading to a third wave in early 2010.

Author:  Siam [ Fri Nov 27, 2009 3:46 pm ]
Post subject:  Re: Recombinomics Commentary


D225G Ukraine Norway Link and China Spread
Recombinomics Commentary 14:15
November 25, 2009

The same mutation has been found in both fatal and mild cases elsewhere, including in Brazil, Japan, Mexico, Ukraine, and the United States, said the WHO.

WHO's spokeswoman in Beijing, Vivian Tan, said the agency is aware of three such cases in China that occurred in June and July that were similar to the cases being investigated in Norway. Tan said WHO had no information on the cases mentioned in the Xinhua report Wednesday.
There is no evidence the mutated swine flu virus is circulating widely in the world, Tan said, but since it has been linked to deaths in Norway and elsewhere, investigators are focusing on whether this mutation could be a marker for more severe disease.

The above comments are associated with a report of eight D225G isolates with in China. Genbank has three of the sequences, as noted earlier. The increase in examples to eight is not surprising since D225G was in four of four fatal cases in Ukraine and reports from Norway cited detection in three isolates, 2 fatal and 1 severe case who had recovered.

However, 25 HA sequences deposited at Genbank had an HA sequence that contained D225G, A/Norway/2924/2009, but also had the wild type sequence, raising concerns that D225G was circulating as a mixture that was most easily detected in lung samples, because the D225G was more than a "marker". It is a polymorphisms that was found in 1918 and 1919 samples which were well characterized for receptor binding properties, which indicated the change conferred increased binding to gal 2,3 receptors which are present in lung.

Concerns that this receptor binding domain change was widely circulating were increased because an HA marker on the Norway isolate with D225G was found on additional isolates in Norway, and worldwide (see list here). This marker was also in the HA sequences from the fatal cases in Ukraine. Full sequences from one of the Norway isolates, A/Norway/3364-2/2009, were deposited, and a marker on the NA sequence matched isolates with the HA marker, linking this broader set of isolate from Norway with the Ukraine sequences, which also had the NA marker, as did multiple other isolates (see list here).

These associations link Ukraine to Norway, but the D225G polymorphism has jumped onto multiple genetic backgrounds, which are widespread, increasing concerns the D225G is circulating undetected because it is concentrated in lung tissues and is poorly represented in nasopharyngeal swabs.

Release of full sequences from China, as well as Norway, would be useful.

Author:  Siam [ Fri Nov 27, 2009 3:47 pm ]
Post subject:  Re: Recombinomics Commentary


Worldwide Transmission of D225G
Recombinomics Commentary 06:45
November 27, 2009

The recently released sequences from patients in Ukraine provided valuable insight into the pathogenicity of H1N1 and the genetic change associated with the total destruction of both lungs in fatal cases. The description of the patients and the spread of receptor binding domain change, D225G, to multiple genetic bakgrounds via recombination led to the prediction that D225G would be found in the lung samples from fatal cases. The release of the sequences by Mill Hill confirmed the prediction. Sequences from 10 isolates were released and all four fatal cases had D225G. Moreover, all 9 cases from western Ukraine, which were from three Oblasts (Ternipol, Lviv, and Khmelnitsy) were from the same sub-clade as the fatal cases, but the samples from the upper respiratory tract did not have D225G. The absence of D225G from the upper respiratory tract is not a surprise because the specificity of D225G included alpha 2,3 receptors which are present in the lungs. Thus, the sub-clade with D225G can expand and cause a cytokine storm which destroys the lungs. Moreover, sequences with D225G have been designated as low reactors by Mill Hill, raising concern that immune responses and vaccine will select for D225G.

Although D225G transmits from patient to patient, only the samples from the fatal cases, which were from lung and throat samples were positive for D225G. The sequences from Ukraine led other countries to more fully investigate samples. Norway, which had seen an increase in fatalities announced the detection of D225G in two fatal and one severe case. Although those sequences have not been released, 25 HA sequences were subsequently released and one sequence had D225G as a mixture, confirming the mixed nature of samples with D225G.

Moreover, the sequence with D225G was the same sub-clade as Ukraine, and several matching sub-clades were in subsequent samples, but those samples did not contain D225G, again pointing to a requirement for sampling of appropriate tissues.

The implication of this sub-clade in the increase in deaths in Norway was the finding that the first fatality was also linked to the same sub-clade. The patient, 43F, was in previously good health and given a prescription for Tamiflu after visiting the hospital. However, she was sent home and died two days later. This type of rapid death had been noted for many of the Ukraine cases. Full sequences were generated confirming that the NA sequence was closely related to the NA sequences from western Ukraine, but the sample was from the trachea and did not have the D225G. However, the association of this sub-clade with the first patients death and the finding of D225G in the first isolate matching this sub-clade support D225G transmission as a mixture, with detection in appropriate sampling of the lower respiratory tract.

Phylogenetic analysis of public sequences indicate that the Norway/Ukraine is widespread, strong suggesting that D225G has sread worldwide. However, detection of D225G, as was seen in Ukraine requires that the proper samples are tested.

The finding of D225G in four of four fatal cases in Ukraine leaves little doubt that the polymorphism is transmitting and the recent classification of Ukraine sequences carrying D225G suggests the spread will accelerate. The finding of the same change in unlinked patients in two Oblasts in western Ukraine is similar to the finding of swine H1N1 in two counties in southern California in April. The fact that the two California cases had no link to swine or each other, and were collected from patients over 100 miles apart conclusively demonstrated that the swine H1N1 was efficiently transmitted human-to-human and many more cases would be identified.

The data from Ukraine conclusively demonstrate that D225G is efficiently transmitting and the transmission traces back to earlier isolates from Norway of the same sub-clade with D225G.

Since D225G is frequently not detected in samples from the upper respiratory tract, another method of tracking is through phylogenetic analysis, which shows that the Norway/Ukraine sub-clade is widespread, even though all HA sequences do not have D225G, as was seen in Ukraine.

The worldwide transmission of the Norway/Ukraine sub-clade, or other sub-clades with D225G raises concerns that associate hospitalizations and fatalities will have a significant uptick as an increasing number of patients get exposed to this sub-clade linked to D225G.

More surveillance of low respiratory tract infections would be useful.

Author:  Siam [ Fri Nov 27, 2009 3:50 pm ]
Post subject:  Re: Recombinomics Commentary


D225G Evades Immune Response
Recombinomics Commentary 08:18
November 27, 2009

Antigenic characterisation: A/California/7/2009 like. Low reactor
The above comment is for the updated characterization sheet for A/Lviv/N6/2009, one of the four isolates from fatal cases in Ukraine. All four isolates had D225G, and in the above isolate that was the only non-synonymous change in HA, indicating the polymorphism was escaping from the immune response.

D225G is in the receptor binding domain and therefore at the surface of HA, and several different polymorphism were noted at that position (D225G, D225E and D225N) suggesting the changes were to escape from the immune response. Moreover the change was appearing on multiple different genetic backgrounds. This is not surprising, since position 225 is in an antigenic site and D225N was linked to the establishment of S31N in the H3N2 population.

However, this low reactor status is in marked contrast to earlier studies on vaccine target clones, which indicated D225G produce no difference in titer, highlighting the vagaries of reference anti-sera data.

This escape could also explain the increases in reported D225G cases as well as increases in hospitalizations and death.

More information on the discrepancy in results from two reference anti-seras would be useful.

Author:  Siam [ Fri Nov 27, 2009 3:54 pm ]
Post subject:  Re: Recombinomics Commentary


WHO Silence on D225G Immune Escape Raises Concerns
Recombinomics Commentary 13:18
November 27, 2009

The recent upgrade of the characterization sheet for A/Lviv/N6/2009 to "low reactor" status has created significant pandemic concern. The change affects receptor binding specificity and allows the virus to bind alpha 2,3 targets which are on the lung, and also affects the antigenic site. However, early data on the development of the attenuated vaccine target indicated that there was no difference between the response to wild type and D225G. These differences have not been explained, although the testing of the candidate vaccine target would be on the cold adapted background, while the testing that produced the "low reactor" designation would be on the D225G on its natural swine H1N1 background. Initial investigations were carried out by Mill Hill, but then the CDC in Atlanta was also involved, possibly to confirm the "low reactor" results.

However, WHO has not issued any updates on this development. They have said that the D225G change in Ukraine was "not significant" even though it was in four of four fatal cases and now has been designated a low reactor. The only indication is at the GISAID database, which is public, but requires membership and is password protected. This designation has serious implications because there is direct and circumstantial evidence that D225G is circulating as a mixture, and immune responses that fail to target D225G can shift the ratio in favor of D225G, which could lead to a significant rise in severe and fatal cases.

It was the rise in severe and fatal cases that led Norway to closely examine cases there, and D225G was found in three cases (2 fatal and 1 severe). However, release of sequences from Norway identified a fourth case, where D225G was identified as a mixture with wild type. Moreover this mixture was the earliest sub-clade in Norway that matched the sub-clade in Ukraine. This sub-clade was isolated prior to the first reported fatal cases in Norway, which was also the same sub-clade, although D225G was not present in the sequence from a throat swab from that patient.

The failure of the WHO or CDC to comment on the low reactor status of the Ukraine sequences from fatal patients is also cause for concern. More detail on this designation, and vaccine plans to address this issue, would be useful.

Author:  Siam [ Fri Nov 27, 2009 3:56 pm ]
Post subject:  Re: Recombinomics Commentary


D225G and H274Y in Fatal Infection in France
Recombinomics Commentary 17:30
November 27, 2009

In a statement, the Institute suggests "Reference of mutations in the genome of influenza virus A-H1N1" from these two people who had no relationship and were hospitalized in two different cities. InVS was that for one of these patients, in addition to this mutation, another known mutation that is resistant to Tamiflu, the drug used to treat people infected with the virus.

The above translation describes two fatal cases in France with D225G. Moreover, one of the two cases also had Tamiflu resistance, presumably H274Y.

The presence of D225G in unrelated patients at distinct locations mirrors the results in southern California in April, when swine H1N1 was initially reported in the United States. The same strain in two patients who had no link to swine or each other signaled efficient transmission. The same is true for D225G. It is in multiple patients in multiple countries and appearing at increasing frequencies at the same time.

The finding of Tamiflu resistance in one of the fatal infections raises additional concerns. The circumstances surround the resistance would be useful. The number of reports of H274Y have spiked in the past week, suggesting it too is efficiently spreading at a detectable level.

Author:  Siam [ Fri Nov 27, 2009 4:09 pm ]
Post subject:  Re: Recombinomics Commentary


WHO Mis-statements in D225G and H274Y Raise Concerns
Recombinomics Commentary 21:31
November 27, 2009
Siam: the bolding in the comments section are mine.

Preliminary tests reveal no significant changes in the pandemic (H1N1) 2009 virus based on investigations of samples taken from patients in Ukraine. Analyses are being performed by two WHO influenza collaborating centres as part of the global influenza surveillance network.
Preliminary genetic sequencing shows that the virus is similar to the virus used for production of the pandemic influenza vaccine, reconfirming the vaccine's efficacy at this time.

The above WHO comments in their latest Ukraine outbreak update are unfortunate. The comments were made after sequences had been generated which showed that four of four fatal cases in Ukraine had the receptor binding domain change D225G. This change had been predicted because it is the type of change expected for the large number of deaths which were linked to the rapid destruction of both lungs. D225G had been identified in 1918 and 1919 lung samples from fatal infections and analysis of the change identified a change in receptor binding specificity, which included alpha 2,3 receptors found on human lung epithelium. The failure of WHO to consider such a change significant raises serious concerns about the agency's credibility and scientific underpinnings.

Moreover position 225 is in one of the known antigenic sites, so to declare a confirmation of vaccine efficiency was false. H1N1 sequences with three changes at position 225 (D225G, D225E, and D225N) had already been reported and D225N on H3N2 was associated with the fixing of S31N (Adamantane resistance) in seasonal H3N2. Thus, a change at that position did not confirm the efficiency of the vaccine and the characterization sheet for one of the four sequences from the fatal cases was just declared a "low reactor" indicating the efficacy of the vaccine was not confirmed and the cause was likely to be D225G since it is the only non-synonymous HA change on the Ukraine sequences from fatal cases.

The failure of WHO to correctly report on the receptor binding domain after the sequences had been generated destroys confidence in the agency at a most crucial time. In addition to targeting of the lungs and a reduction in vaccine efficiency,Tamiflu resistance is on the rise and one of the fatal D225G cases in France also has H274Y, raising concerns at a time when WHO is posting situation updates which are not credible.

They then compound this lack or credibility by claiming that the D225G, which is four unrelated cases in Western Ukraine are "spontaneous" which is also the characterization of oseltamivir resistance, H274Y. These claims have no scientific basis and are simply absurd.

These statements by WHO, at a time of extremely peril, are both scandalous and hazardous to the world's health.

Author:  rutsuyasun [ Fri Nov 27, 2009 9:49 pm ]
Post subject:  Re: Recombinomics Commentary

Emphases are mine.

WHO Confirms D225G Vaccine Failure
Recombinomics Commentary 03:31
November 28, 2009

One isolate from Ukraine with the mutation had changed so that swine flu vaccine probably would not protect against it well, Britain's national medical laboratory reported Friday.

Flus mutate so fast, Dr. Fukuda cautioned, that announcing each change is "like reporting changes in the weather."

The above quote from tomorrow's NY Times piece by Donald McNeil, acknowledges the vaccine failure for viruses with D225G. However, although WHO has publicly confirmed the failure, they don't think an announcement is required. Thus, they continue to offer altering opinions on the significance of D225G, which directs H1N1 to the lung and was present in four of four fatalities in Ukraine.

The associate of D225G with the Ukraine fatalities led to a survey of samples in Norway, where D225G was found in three patients (two who died and 1 who was in serious condition). Similarly, France found D225G in two fatal infections, including one who was Tamiflu resistant.

However, even though this change is drawing additional attention daily, WHO has taken a position that the vaccine failure against H1N1 with this D225G is not worthy of an announcement.

This mindset is significant cause for concern and is hazardous to the world's health. ... G_Vac.html

Author:  Siam [ Sat Nov 28, 2009 5:22 pm ]
Post subject:  Re: Recombinomics Commentary

WHO Silence on D225G Vaccine Mismatch Confuses Media
Recombinomics Commentary 15:53
November 28, 2009

The vaccine can still prevent the virus from entering body cells to reproduce. These new changes should instead encourage all French to go to be vaccinated with adjuvant.

The above translation is from a French story on the discovery of D225G in two fatal French cases, one of which was also Tamiflu resistant. Because of the failure of WHO to disclose the "low reactor" status of Ukraine H1N1 with D225G, media reports are distributing the false information, stating that the activity of the vaccine is unaffected by D225G.

This misinformation is fueled by the WHO update on Ukraine, which stated that the vaccine appeared to be unaffected based on the sequence. For the "low reactor" the HA only had one amino acid difference, which was D225G.

As has been noted daily in new media reports, D225G is widespread, and phylogenetic analysis shows that the sub-clade in Norway and Ukraine is even more widespread than reported isolate with D225G, raising concerns that the D225G is circulating undetected because most samples are collected from the upper respiratory tract, while D225G is largely in the low respiratory tract.

The designation of a "low reactor" means that the titer of a reference anti-sera is reduced by at least 4 four. A four fold reduction in titer is typically called a mis-match and mismatched vaccines are a concern because a vaccination will not eliminate the new changes that reduced the titer, but will eliminate the wild type that competes with the variant. Therefore the use of a poorly match vaccine leads to increased vaccine resistance and in this case would select for D225G.

The failure of WHO to address this issue is the height of irresponsibility. Although the NY Times noted the Ukraine low reactivity due to D225G, it is not clear that the WHO comments were in response to the vaccine failure, since similar statements were made at the WHO virtual press conference and the reactivity of the vaccine was not addressed in statements or answers to questions, although the weatherman comment was in responces to questions about D225G..

Therefore a statement by WHO is long overdue to end that false information distributed through media reports and the WHO needs to address plans to deal with the vaccine failure against D225G and potential similar failures against D225E and D225N which are also widespread.

Author:  Siam [ Sat Nov 28, 2009 5:25 pm ]
Post subject:  Re: Recombinomics Commentary


WHO Weather Report on Ukraine H1N1 D225G Required

Recombinomics Commentary 17:13
November 28, 2009 ... ather.html

The question is whether this mutation again suggests that there is a fundamental change going on in viruses out there or whether there is a turn for the worse in terms of the severity. I think that the answer right now is that we are not sure. I want to answer why we are not sure in a way which explains why more investigations are needed. As you know these influenza viruses change frequently. Their gene properties change because these are viruses which frequently undergo mutations and so mutations in and of themselves are not necessarily important and in fact, if every mutation was reported out there, it would be like reporting changes in the weather -

The above comments from Keiji Fukuda at Thursday's virtual press conference in context, which highlights the need for additional data on D225G. However there is a large body of evidence on this change and the Ukraine data is the most current and quite compelling. Ten samples were collected from ten patients and nine represented the same sub-clade. However, only four were from tissues from fatal cases, but all four had D225G. In contrast, nasopharyngeal washes were collected from the five surviving patients in western Ukraine and all sequences were the same sub-clade, but did not have D225G. The finding of D225G in 100% of the fatal cases raises concerns, but the number of such cases is small. However, well over 400 people have died in Ukraine, so additional samples from fatal cases should not be a problem, and collection of samples from both upper and respiratory tracts in the same patient would be useful.

An aggressive campaign on this change is warranted because the only reported tested sample for antigenicity was found to be a low reactor, and the only amino acid change in the HA sequence was D225G. Since the same sub-clade was found in earlier collections in Norway, and the cases positive forD225G were fatal or severe, more interest in the change has been generated, but media reports state that the vaccine is effective against D225G, when the data presented on the Mill Hill characterization sheet at GISAID cites the antigenicity as being "low reactor", signaling a need for a new vaccine.

It has now been a month since the reported Ukraine samples were collected and hundreds have died there in the past month, so additional sequence data on fatal cases, including upper and lower respiratory tract origins from the same patient should be available at this time.

Therefore, release of these sequences and a statement on the initial results, including the low reactor status of isolates from fatal cases in Ukraine would be useful. Finding a receptor binding domain change which alters specificity in 100% of fatal cases is cause for concern, and release of additional sequence data is overdue, since some still need a weatherman to know which way the wind is blowing.

Author:  Siam [ Sat Nov 28, 2009 6:23 pm ]
Post subject:  Re: Recombinomics Commentary

Week 46 Sets Record H1N1 US Pediatric Fatalities
Recombinomics Commentary 17:13
November 28, 2009

There were 35 influenza associated pediatric deaths reported week 46: NH (1), MA (1), RI (2), PA (2), MN (1), MO (1), NC (2), FL (3), TN (1), TX (2), CO (1), NM (8), WA (1), CA (1), IL (3), IN (1), KY (1), NY (1), SC (2)

The above description of the 35 fatal pediatric deaths in week 46 in the US will be published on Monday. The 35 ties that daily record set two weeks ago, and the new entries raise the 2009/2010 season total to 178, which is a new record. The 138 last week represented the highest tally since adolescent fatalities became mandatory, when 142 died in 2003/2004. 178 is a new all time high, but it will be broken each week until there is a break, which may not happen in 2010.

The pediatric deaths allow for a true comparison of swine flu to seasonal flu, because the tally represents lab confirmed cases for both, in contrast to the much cited 36,000 seasonal flu casualties which is an estimate and largely (90%) represents fatalities who are over 65.

However, week 46 is still the beginning of the traditional flu season, and since the start of mandatory reporting, the first reported fatality for the season is in January, so the season total for week 46 has always been zero.

Author:  Siam [ Sun Nov 29, 2009 11:03 am ]
Post subject:  Re: Recombinomics Commentary

Siam: Mill Hill (a WHO coordinating lab), the report on the Ukranian sequence with D225G which they published (referenced by Niman in this Commentary) details that this mutation engendered a "low reactor."

That's against the vax target, A/California/7/2009, specifically.

The vax response to the mutated virus with D225G found in the fatal Ukranian strains is not optimal.

H1N1 RBD Changes at 225 Create Vaccine Mismatch Concerns

Recombinomics Commentary 14:22
November 29, 2009 ... 5_Mis.html

Originating Laboratory provider of clinical specimen(s)
and/or virus isolate(s)
[Ukraine, Kiev] Ministry of Health of Ukraine

Central Sanitary Epidemiological Station 41 Yaroslavskaya str. 04071 Kiev Ukraine

Sample ID given by the sample provider

Submitting Laboratory generator of data
[UK, London] WHO Collaborating Centre

The National Institute for Medical Research (NIMR) The Ridgeway - Mill Hill London NW7 1AA, UK

Sample ID given by the sequencing lab:

Antigenic characterisation:
A/California/7/2009 like. Low reactor

The above characterization of A/Lviv/N6/2009, which was placed on deposit at GISAID by Mill Hill, raises concerns about the evasion of pandemic H1N1 sequences which change position 225. The above isolate has only one amino acid change in HA, D225G, which strongly implicates D225G in the low reactor results. A low reactor reduces the titer by four fold or more, which signals a mismatch. Mismatched vaccine create the potential for the section of the variant, which could create problems since D225G was found in four of four fatal cases in Ukraine, and several countries (Brazil, Ukraine, Norway, France, China) found D225G in fatal and or severe cases.

Moreover, since D225G changes receptor specificity, it may transmit undetected because of an emphasis on nasal pharyngeal samples where levels would be expected to be lower. This was seen in five additional nasopharyngeal washes from Ukraine survivors, who were infected with a sub-clade that matched the fatal cases, but lacked the D225G.

Similarly, the first matching sub-clade isolate in Norway was a mixture of D225G and wild type. Public sequences from subsequent sub-clades were negative, but the sequence from the first reported fatal cases appears to be one of multiple collections, and the WHO briefing on the situation in Norway stated that D225G was detected in the first fatal cases, suggesting this case was also infected with a mixture. The ratio of D225G to wild type would increase under vaccine selection pressure, as indicated by the "low reactor" status of the isolate above. Although the sequence databases under-represent the transmitting levels of D225G, the positive samples have been collected throughout the world, and these levels may be increasing.

Moreover, position 225 has undergone additional changes in pandemic H1N1. D225E and D225N have been detected worldwide also raising concerns that these isolates will also be low reactors and also increase because immunological escape. Similarly, mixed signals have been seen for D225E and multiple codons encode D225E, suggesting it too may be selected by immune response. Since most of these isolates were collected prior to the distribution of the exiting vaccine, it is likely that selection was being driven by host's immune e response.

More information on the antigenic characterization of additional receptor binding domain isolates, especial those that alter postion 225, would be useful.

Author:  Siam [ Sun Nov 29, 2009 3:05 pm ]
Post subject:  Re: Recombinomics Commentary

Niman states at PFI: The bottom line is pretty simple. Mill Hill tested one of the isolates from a fatal case in Ukraine. The HA protein sequence had one change, D225G, and the antigenic designation for the virus was "low reactor". That means the the D225G knocked down the titer by at least four fold.

Vaccine effectiveness is measure by how many vaccinated volunteers have a titer rof 40 or more. The 40 cut-off is pretty much borderline. Although some with a titer of 40 will be protected from infection, other will not. D225G will knock that number down at least four fold, so those with titers of 40 or 80 won't be protected and 160 will be borderline. However, the sheet just said "low reactor" so the reduction could be more than four fold.

Bottom line is a low reactor is a mismatch amd mismatches are bad ideas because not only can you be infected, but you can kill off the wildtype and make the infection closer to pure D225G if it startes as a mixture. D225G goes to the lungs where there can be major problems.

Moreover, transmisison may involve a much high ratio of D225G.

This selection presure is not only for the vaccine. It also applies to "natural" immunity in those infected previously by wild type.

Thus, the same scenario may be playing out for D225E and D225N, since position 225 is an antigenic site.

So the bottom line is a low reactor with a change at position 225 is a bad situation, while WHO and CDC are saying NOTHING with regard to the vaccine

Author:  Siam [ Mon Nov 30, 2009 3:40 pm ]
Post subject:  Re: Recombinomics Commentary


H1N1 Evolution Outpacing Vaccine and Host Defenses

Recombinomics Commentary 13:32
November 30, 2009 ... ution.html

The designation of one of the Ukraine D225G isolates as a "low reactor" raises concerns that the H1N1 evolution is outpacing the vaccine as well as immune responses from unvaccinated hosts. This concern was present ealry when changes began to appear at position 225, a known antigenic site.

One of the highest profile isolates was the San Francisco traveler to Hong Kong, who was reported (in July) to have Tamiflu resistant H1N1, even though she had not taken Tamiflu. Her case was mild and she recovered without antiviral treatment, but the virus had a receptor binding domain change, D225E. A survey of D225E at the time demonstrated that it was first reported in the US in New Jersey, but not subsequently. However, it had spread to many other countries, raising concerns that the US surveillance was missing this widespread sub-clade.

Moreover, there were soon examples of D225G and D225N in other early samples supporting the use of position 225 to escape host defenses. The concern of multiple changes at the same position increased when sequences from fatal cases in Sao Paulo were releasd recently. The July/August collections had D225G in two of two fatal cases, and D225N in two of two other fatal cases, with 3 of 4 samples coming from lung.

These concerns increased when sequence data from Ukraine was released and 4 of 4 fatal cases had D225G.

The above data reinforced concerns that the changes at position 225 were becoming more prevalent, especially in samples collected from affected organs of fatal cases.

The concerns ratcheted up another notch when of the four cases was characterized antigenically, and was reported to be a "low reactor". Since the low reactor had only one amino acid change in HA, the role of D225G in the reduced titers raised the level of concern higher.

However, WHO has yet to address this result. The "low reactor" status was quietly added to the characterization sheet at GISAID and there has been little comment in the mainstream media, other than a sentence in the New York Times on Friday.

Comment form WHO and other agencies, including Mill Hill and the CDC, who were conducting testing of the Ukrainian samples is long overdue.

Author:  Siam [ Tue Dec 01, 2009 10:33 pm ]
Post subject:  Re: Recombinomics Commentary ... f=5&t=3389

Dr Niman wrote this over at flutracker/ hat tip/thxs/

Author Message

Post subject: Mill Hill Designates Ukraine with D225G a Low

ReactorPosted: Fri Nov 27, 2009 2:11 am

Location: Pittsburgh, PA USA Originating Laboratory
provider of clinical specimen(s)
and/or virus isolate(s) [Ukraine, Kiev] Ministry of Health of Ukraine
Address: Central Sanitary Epidemiological Station 41 Yaroslavskaya str. 04071 Kiev Ukraine
Sample ID given by the sample provider
Submitting Laboratory
generator of data [UK, London] WHO Collaborating Centre
Address: The National Institute for Medical Research (NIMR) The Ridgeway - Mill Hill London NW7 1AA, UK
Sample ID given by the sequencing lab:
Antigenic characterisation: A/California/7/2009 like. Low reactor




Location: Pittsburgh, PA USA Isolate ID: EPI_ISL_62012
Isolate name: A/Lviv/N6/2009
Passage details/history: throat swab
Type: A / H1N1
Lineage: swl

The above isolate has been designated a low reactor by Mill Hill.




Location: Pittsburgh, PA USA niman wrote:
Isolate ID: EPI_ISL_62012
Isolate name: A/Lviv/N6/2009
Passage details/history: throat swab
Type: A / H1N1
Lineage: swl

The above isolate has been designated a low reactor by Mill Hill.

The only protein change on the HA of the above sequence is D225G.



can someone explain the significance?
this is important somehow. How?



Post subject: Re: Mill Hill Designates Ukraine with D225G a Low ReactorPosted: Fri Nov 27, 2009 2:40 am

This would fall into the EXTREMELY bad news category. It means that not only does D225G drive the H1N1 to the lungs, but it allows the virus to evade the natural immune response / vaccine.

Author:  Genesis [ Wed Dec 02, 2009 7:09 am ]
Post subject:  Re: Recombinomics Commentary

thank you for the post/link siam. looks like it might be getting to be quite serious, isn't it.

i took your link. niman has posted something after:

The virus still seems to be a mixture, so even though the prior exposures may select for D225G, your existing immunity may knock down the wild type and then allow for more focus on D225G. Moreover, not all H1N1 infections involve D225G.

i take it this means anyone having had any of the current versions of H1N1 should impart some immunity advantage to this more serious version with D225G. somewhere i read where this change is an internal element of the virus, not the protective protein of the outside layer, which theoritically should mean prior exposure resulting in antibodies should provide what i hope is quite a bit of immunity. the antibodies will still have the usual defence mechanism attacking the outside of the virus.

i guess this doesn't quite agree then with what niman implies? :dunno

i'll see if i can find a link in my history, but i bet it's been too long ago. :embarressed

Author:  Siam [ Fri Dec 04, 2009 11:29 am ]
Post subject:  Re: Recombinomics Commentary


446 Dead in Ukraine - D225G and Deaths Spread
Recombinomics Commentary 02:20
December 2, 2009

1,937,292 Influenza/ARI

116,982 Hospitalized

446 Dead

The above numbers represent the latest update from the Ukraine Ministry of Health. As the number of cases approach 2 million, the rate of increase has slowed and shifted to the east. The largest daily update was in Donetsk, which rose by 5892 cases to 127,584 (see map). However. Increases in deaths are being report across the eastern border in Russia, were there have been 30 deaths reported in Saratov. Similarly, almost 200 deaths have been reported in Turkey, raising concerns of D225G spread.

The initial data showed strong association of D225G with fatal cases. Of the 9 samples from western Ukraine, all four fatal cases had D225G. In contrast nasal washes from surviving cases were positive for the same sub-clade, but were wild type at position 225, suggesting that infections were mixtures and virus with D225G targeted the lung, while wild type remained in the upper respiratory tract.

This tissue tropism poses surveillance problems, because most sequences come from nasal pharyngeal swabs, which may not reflect D228G in the lung. Currently, only 1% of HA sequences have D225G, although it was present in April in the vaccine target, A/California/7/2009, as a mixture. The version with D225G was selected for the target in the attenuated carrier distributed as a flu mist. In contrast, the killed infected vaccine has the HA version of California/7 with wild type D at position 225, raising concerns that killed vaccine or natural immunity will select for D225G. This concern was increased when Mill Hill labeled Ukraine H1N1 with D225G a low reactor.

The reporting of D225G in all fatal cases in Ukraine should be followed up with additional sequence data on more of the 400+ fatalities. Collections from the upper and lower respiratory tract in fatal cases would help define the significance of D225G in fatal lung cases, and also determine the ratio of components of mixtures with and without D225G.

An increase in the D225G ratio would be cause for concern

Author:  Siam [ Fri Dec 04, 2009 11:42 am ]
Post subject:  Re: Recombinomics Commentary


H1N1 Tamiflu Resistance Crosses Tipping Point
Recombinomics Commentary 03:07
December 4, 2009

Within the past two weeks, the number of documented cases of oseltamivir resistance in H1N1 viruses has risen from 57 to 96. Around one third of these cases occurred in patients whose immune systems were severely suppressed by haematological malignancy, aggressive chemotherapy for cancer, or post-transplant treatment. The clusters in the two hospital wards should be viewed in the context of these overall trends. Although all incidents of oseltamivir resistance merit investigation, no evidence suggests that events to date constitute a public health threat.

The above comments from the WHO update on transmission of H1N1 with H274Y in hospitals in Wales and North Carolina, claims no evidence suggests that the sharp spike in cases, or the clear human to human transmission, is a public health threat, but the evidence in the report alone clearly identifies a threat. Moreover, the numbers in the report appear to represent only a subset of associated evidence, which raises concerns that a tipping point has been reached and readily detectable H274Y will be reported in the upcoming weeks, signaling the early stages of fixing H274Y in pandemic H1N1, as happened in seasonal H1N1.

The rapid appearance of H274Y in patients receiving Tamiflu suggested that H274Y was transmitting as a minor species and was largely undetected in untreated cases. However, there was concern that the H274Y level would rise, leading to readily detected transmission. The reports of transmission in clusters of immuno-suppressed patients indicate the concern has been realized, and transmission resembles the early stages of fixing of H274Y in H1N1 seasonal flu.

In addition to the clusters in Wales and North Carolina, smaller clusters have been reported and recent sequences show that H274Y in earlier sub-clades is appearing more frequently, which represents fit sub-clades.

The sub-claded linked first examples of H274Y in a patient not receiving Tamiflu has now also been reported in an H274Y case in Tennessee, while recent cases in Georgia and Spain represent another sub-clade with multiple examples of H274Y. The lack of linkage in these examples suggests that the level of H274Y is much wider than reported, which may be linked to limited testing of milder cases.

Thus, the explosion of cases with H274Y is expected to continue to rise and create more problems in the management of patients. H274Y will eliminate Tamiflu and Peramivir and limit anti-virals to Relenza, which will further strain the health care system.

Siam: going on personal experince with this flu, the third day, without Tamiflu, I would have been in Hospital. On the second day, it hit my chest, hard. This means the medical system would be very quickly overrun.

Tip: I am only posting the really interesting and important commentaries, if you just read them, ignore the stuff you don't understand, shortly you will be understanding his comments alot better.

Author:  Siam [ Sat Dec 05, 2009 7:02 pm ]
Post subject:  Re: Recombinomics Commentary

WHO in Denial on Tamiflu Resistance and RBD Changes

Recombinomics Commentary 21:21
December 5, 2009

In the USA outbreak, which involved four severely immuno-compromised patients, cases occurred in a two-week period between mid-October and early November. Three of the four cases were fatal, but the role of H1N1 infection in contributing to these deaths is uncertain.

The above comments are from the WHO update on transmission of H274Y are hospitals in the United States and United Kingdom. As indicated above, the virus was not only was fit enough to transmit human to human, but also lead to fatal infections in the majority of those infected. Although these patients had underlying conditions, the death of three of four demonstrates that the virus is quite virulent.

However, as was seen in comments on the receptor binding domain change in Ukraine which was detected in all four fatal cases, these dramatic developments are discounted in WHO reports. The outbreaks in hospitals are characterized as being somewhat different from the general population. However, although these patients are more likely to develop serious illness, the majority of the human population is naïve for this virus, so initial infection rates would be similar. The immuno-compromised patients are monitored ore closely, so detection in these patients would be increased, but they would be representative of initial infections.

Like the general population, the reports of cases of H274Y have increased dramatically in the past few weeks, how, in contrast to what is implied in the report, is the rate of detection that is alarming. In the US, that rate has increased almost 10 fold in the past few weeks.

Thus, the WHO report is really a denial of the actual resistance situation. The cases in the hospitals are representative of initial infections in the general population and the rate of infection has spike higher leading tyo a large number of new cases, even though a lower number of samples are tested.

The denial of the explosion of H274Y as well as the association of D225G in fatal lung infections raises concerns that these events are being portrayed in an overly optimistic light. Although cases are declining in many parts of the world, these genetic changes in transmission and ant-viral resistance raise concerns that the next wave will be markedly more virulent and difficult to treat at a time when the efficiency of anti-virals and the utility of the vaccine target are in a decline. ... enial.html

Author:  Siam [ Sat Dec 05, 2009 7:29 pm ]
Post subject:  Re: Recombinomics Commentary


H1N1 Tamiflu Resistant Cluster in Pennsylvania
Recombinomics Commentary 17:40
December 5, 2009

"They've detected two cases in the state, in the eastern part of the state, and I don't think that's unexpected. We're seeing growing numbers in the country of resistant viruses showing up, and we know flu mutates quite readily," Dr. Dixon continues.

The above comments describe another Tamiflu resistant cluster in the US. It is not clear how closely linked these cases are, but the report follows a cluster in the Maryland/Virginia area, as well as human to human transmission at Duke, where three of the four cancer patients died.

These clusters of cases likely represent a very small percentage of the resistance transmitting in the US and worldwide. Recently WHO noted an increase fro 57 to 96 of reported cases in the past 2 weeks and CDC weekly reports show an increase in frequency of almost 10 fold (12/464 tested samples in the past 3 weeks vs 3/1076 in the prior weeks of the current 2009/2010 season).

In addition, the CDC has released sequences with H274Y from Tennessee, Georgia, and Tanzania, providing additional evidence on the spread of the polymorphisms.

These repeated reports of H274Y in geographic clusters, as well as transmission clusters, suggest that H274Y has passed a tipping point, and reports of H274Y will increase in the upcoming weeks.

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