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 Recombinomics Commentary 
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Post Re: Recombinomics Commentary
WHO in Denial on Tamiflu Resistance and RBD Changes
Recombinomics Commentary 21:21
December 5, 2009

http://www.recombinomics.com/News/12050 ... enial.html

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.

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Post Re: Recombinomics Commentary
D225G and D225N In Utah Patient Raises Pandemic Concerns
Recombinomics Commentary 23:55
December 6, 2009

http://www.recombinomics.com/News/12060 ... _Utah.html

The CDC has put HA sequences, A/Utah/42/2009, from a Utah patient (28F) on deposit at Genbank, which has D225G and D225N. Moreover, the sequence from the original sample gives mixed signals at tandem positions, revealing significant heterogeneity that the codon. A clone for the original sample has D225G. D225N and D225G have been identified in necropsy lung samples from Brazil (A Sao Paulo/53845/2009 and A/Sao Paulo/53838/2009 have D225N while A/Sao Paulo/53225/2009 and A/Sao Paulo/53206/2009 have D225G) raising concerns that these polymorphisms are associated with more severe disease.

The severity may be linked to viral load, because D225G and D225N were present as mixtures in early cases in the US in California and Texas. In fact the vaccine target, A/California/7/2009 was a mixture for D225G, as were several other isolates isolated at about the same time in Texas and California. Other isolates in the same areas, as well as New York, did not give mixed signals, but D225G was found in milder cases.

More recently, these changes were found in more severe cases. All four fatal cases in Ukraine had D225G, while the first fatal cases in Norway had D225G and D225E SNPs. The Utah case above however is the first sample with D225G and D225N. In the United States, the two isolates in New York with D225N had an additional polymorphism which was also in A/Ternopil/N11/2009, a fatal case from Ukraine, suggesting additional relationships which are not well represented in the existing database. These relationships may be obscure because of tissue specific expression, and mixtures which are impacted by cloning procedures.

However, the recent findings sequences encoding D225N and D225G, as well as wild type, in the same sample raises concerns that this diversity has paved the way for rapid and varied responses to immunological pressures which has already cause concerns due to the low reactor status of D225G when tested against anti-sera directed against the target of the killed vaccine.

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Post Re: Recombinomics Commentary
Ukraine Cases Spike - D225G and D225N Found
Recombinomics Commentary 23:55
December 8, 2009

2,187,836 Influenza .ARI

132,178 Hospitalized

472 Dead


The above numbers, from the Ukraine Ministry of Health represent a spike in cases yielding a daily increase of 86,578. The jump was widespread, but the biggest jumps were Donetsk increased 12,586 to 160,212 and Dnipropetrovsk which increased 9.813 to 149,358 (see map). This jump may be signaling a move into the real winter season.

Ukraine is under close scrutiny because sequences released by Mill Hill at GISAID had D225G in all four fatal cases and CDC sequences at GISAID identified D225N in new cases, which were also likely fatal, so all HA sequences from fatal cases in Ukraine have had a receptor binding domain change at position 225 (D changing to either G or N). Since the same changes had been seen previously in Sao Paulo, Brazil over the summer, and now cases were being identified in severe and fatal Scandinavian countries,

The finding D225G or D225N in all fatal cases in Ukraine clearly demonstrates that the changes are transmitting, comments from Scandinavian officials notwithstanding. More detail on the new patients and sequences would be useful.

http://www.recombinomics.com/News/12080 ... D225N.html

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Post Re: Recombinomics Commentary
Fatal Tamiflu Resistant H1N1 Cases In the Netherlands

Recombinomics Commentary 13:55
December 8, 2009
http://www.recombinomics.com/News/12080 ... lands.html

A third person that the resistant variant of the Mexican flu had been deceased. This confirms a spokesman for the National Institute of Public Health and Environment (RIVM) Tuesday.. The victim, a woman, was already seriously ill when the flu struck.

A spokesman for the Public Health Service Heart Brabant reports that in one hospital in the region a woman who is already ill with the deceased Mexican flu. The other two deceased patients with the resistant variant had been seriously ill before they received the Mexican flu. In four others were also resistant forms of the Mexican flu found.


The above translation describes three H274Y fatalities, as well as four additional cases in the Netherlands. These data extend numbers released last week which indicated a Tamiflu resistance tipping point had been crossed. In the past three weeks the rate of reported H274Y cases in the US increased almost 10 fold, and human to human transmission had been reported at Duke University hospital as well as a hospital in Wales. Other geographic clusters had been reported in the Washington, DC area as well as eastern Pennsylvania in the US, and Edinburgh in Scotland. This explosion in Tamiflu/Peramivir resistance led to concerns of H274Y pairing up with receptor binding domain changes at position 225.

This type of pairings had been seen in seasonal flu. H274Y had jumped from one H1N1 background to another (Clade 2C, Clade 1, Clade 2B) in the absence of Tamiflu treatment. Although H274Y had jumped from background to background within each sub-clade, the movement in Clade 2B was most extensive and when compared with a receptor binding domain change, A193T, the resistance was fixed in H1N1 season flu.

Similar events were seen in H3N2 seasonal flu and the fixing of S31N, Those events centered on two receptor binding domain changes, S193F and D225N.

Consequently, when the first pandemic H1N1 with H274Y and no linkage to Tamiflu treatment was on an HA with D225E there was concerned that the receptor binding domain change would be linked to a more fit swine H1N1 that would transmit in the absence of Tamiflu. These concerns were increased when another isolate in Tennessee had H274Y and D225E.

However, of even greater concern was the linkage of H274Y with D225G because of the associated receptor binding domain change that drove virus to the lower lung and showed resistance to the swine flu vaccine, as seen for one of the Ukraine isolates with H225G.

These concerns were realized with reports out of France which described two fatalities with D225G and one also had H274Y. H274Y in fatal cases is a significant concern, and three of the four cases at Duke died.

The current case in the Netherlands extends the number of fatal cases with H274Y and raises more concerns on linkage between H274Y and D225G.

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Post Re: Recombinomics Commentary
Siam - it's almost like watching Indonesia all over again, isn't it?

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Wed Dec 09, 2009 7:18 am
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Post Re: Recombinomics Commentary
Yes it is, I guess that is why it hit me so hard, history repeating itself.

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Post Re: Recombinomics Commentary
Commentary

German H1N1Tamiflu Resistance Cluster Raises Concerns

Recombinomics Commentary 17:34
December 20, 2009

For the first time in Germany when swine flu patients - a ten year old and a 31-year-old man - resistance to the influenza drug Tamiflu has been demonstrated.

Both had come with pre-existing conditions and a "weak defense" to the University Hospital of Munster, said the clinic on Friday. They were already infected with swine flu in the reception.


The above translation describes the first to H274Y confirmed cases in Germany. Although Germany has a strong surveillance system and has reported H1N1 since the spring, the first reported cases of H274Y come at a time when reported cases are exploding in the United States, Japan, and the Netherlands, suggesting H274Y is transmitting. This is further supported by the presence of H274Y in two patients from the same hospital. Hospital outbreaks have been reported in Wales and North Carolina in immuno-compromised patients and in North Carolina, three of the four died.

The sudden appearance of H274Y in Germany supports the phylogenetic analysis of recently released sequences from the US and Japan. This analysis shows clustering of H274Y confirmed sequences, and suggests other sequences which are in the same sub-clades also have undetected H274Y.

In one of these clusters, the H274Y is associated with D225E, raising concerns linkage between H274Y and receptor binding domain changes. In another cluster, one isolate has D225G paired up with H274Y, raising concerns that H274Y could be paired with RBD changes linked to fatal or more severe cases.

Thus, the two cases in Germany suggest H274Y is widespread and transmitting, and in some circumstances, the widespread appearance of H274Y may be linked to receptor binding domain changes

http://www.recombinomics.com/News/12200 ... rmany.html

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Post Re: Recombinomics Commentary
Commentary

Emergence and Evolution of Pandemic Tamiflu Resistance

Recombinomics Commentary 12:33
December 22, 2009
http://www.recombinomics.com/News/12220 ... volve.html

The recently pandemic H1N1 sequences from Japan, coupled with earlier sequences with H274Y produce a phylogenetic analysis which clearly demonstrates that the evolution of H274Y in pandemic H1N1 is following the same evolutionary path as H274Y in seasonal flu. In 2008 the Brisbane/59/2007 strain (clade 2B), H274Y went from 0% in some countries like South Korea at the beginning of the year (2007/2008 season) to 100% at the end of the year (2008/2009 season). Moreover, H274Y went to 100% in clade 2B, which represented the vast majority of H1N1 in circulation since the 2008/2009 season, although this year it is being crowded out by pandemic H1N1.

In seasonal H1N1 the role of H274Y was largely that of a hitchhiker. It moved from one H1N1 genetic background to another via recombination, and eventually paired up with the receptor binding domain change, A193T, which led to increases in H274Y in the summer of 2008 in the southern hemisphere and dominance in the fall in the northern hemisphere. In 2008/2009 virtually all Brisbane H1N1 had A193T and H274Y, but there were multiple variation with changes flanking A193T at positions 187, 189, and 196. These acquisitions, as well as early acquisitions, were drive by recombination and most newly acquired polymorphism were acquired from the co-circulating clade 2C (Hong Kong/2652/2006).

The evolution of H274Y began earlier and was characterized by its presence in patients who had not received Tamiflu, demonstrating that the H1N1 with H274Y was evolutionarily fit. The first examples were in the summer of 2006 in China and involved clade 2C. In the 2006/2007, H274Y jumped to clade 1 (New Caledonia/20/1999), but was relative rare in the US and UK, although most positives were in patients not taking Tamiflu. In 2007/2008 the frequency began to rise in clade 2B, which had displaced clade 1 and clade 2A (Solomon Islands/3/2006). The frequency varied, but the highest frequencies were in countries which had one particular clade 2B sub-clade, which was present in 70% of the H1N1 in Norway. The discovery of H274Y in 70% of H1N1 samples in Norway led to widespread screening. H274Y was found in multiple clade 2B sub-clades, but one sub-clade dominated and it acquired additional changes from clade 2C via recombination, including A193T.

This sub-clade was established in South Africa and Australia in the summer of 2008, which set the stage for emergence in the fall of 2008, when H274Y levels in H1N1 approached 100%. This high frequency in seasonal H1N1 led to concerns that H274Y would then jump to pandemic 2009, which emerged in the spring of 2009. Some expected the acquisition to be via reassortment, where pandemic H1N1 would acquire the Brisbane/59 N1. However, there have been no reports of acquisition of a human N1 or any other human flu gene. The pandemic H1N1 has maintained its original constellation of gene segments, with one human (PB1), two avian (PA and PB2) and 5 swine. However, H274Y has jumped from seasonal to pandemic H1N1 via recombination and is currently jumping from one pandemic genetic background to another.

Although H274Y is following the same evolutionary path in pandemic H1N1, the close monitoring of patients, especially those receiving Tamiflu, has led to early detection in patients receiving prophylactic Tamiflu because of linkage to an H1N1 confirmed contact, or therapeutic Tamiflu because of an immune-compromised state. The initial cases were in contracts being treated prophylactically, which led to speculation by Roche that the resistance was "spontaneous" and similar to previously described resistance in children in Japan. However, those earlier examples were linked to sub-optimal dosing and largely limited to H3N2. The resistant H3N2 had changes at multiple positions and did not transmit. Consequently, the switch to optimal doing eliminated the resistance, and there have been no recent examples of H3N2 Tamiflu resistance in Japan, or anywhere else In the world. In contrast, Tamiflu resistance in H1N1 was limited to a single nucleotide which produced H274Y. That same nucleotide change was in H274Y in H5N1 as well as all seasonal H1N1 clades (clade 1, 2B, and 2C) as well as pandemic H1N1. Moreover, the pandemic H1N1 patients receiving prophylactic Tamiflu developed symptoms on day 5 which was close to the normal incubation period of 2-4 days, indicating the H274Y was from a co-circulating H1N1 with H274Y and not due to a spontaneous or de novo mutation. The rapid appearance of H274Y was also noted in patients receiving Tamiflu treatments. In the first case in Singapore, the initial sample was wild type, but two days later H274Y was dominant.

However, the H274Y example that was most analogous to seasonal H1N1 was in patients who had not received Tamiflu. The first example of an evolutionarily fit H274Y was found in a San Francisco traveler to Hong Kong. The June isolate, A/Hong Kong/2369/2009, was a pandemic H1N1 that had also acquired a receptor binding domain change, D225E. Although the initial attempts to find the subclade in northern California failed, the recently released sequences from Japan have an August isolate, A/SHIGA/43/2009, has H274Y and D225E as well as additional HA and NA markers linking the two isolates to the same sub-clade. Moreover, a September isolate from Tennessee, A/ Tennessee/17/2009, also has H274Y with the same series of markers, indicating the sub-clade is widely transmitted over an extended time frame.

In addition to the above sub-clade, additional clustering of H274Y has been noted. One sub-clade includes the isolates from immuno-suppressed patients in Seattle, Washington, as well as the isolate from Illinois, A/Illinois/10/2009, which has also acquired another receptor binding domain change, D225G. This combination of H274Y with D225G has also been described for a fatal case in France, but the sequences have not been made public.

However, the multiple examples of clustering of H274Y in a series of pandemic H1N1 sub-clades demonstrates that H274Y in pandemic H1N1 is following the same path as H274Y in seasonal flu. It has appeared on multiple pandemic H1N1 sub-clades and has also paired up wuth two receptor binding domain changes, D225E and D225G.

Moreover, recently released data from Vietnam has described evolutionarily fit H1N1 in Vietnam in July, leading to infection of 7 healthy students on a train. Similarly, outbreaks of Tamiflu resistant H1N1 have been reported in hospitals in Wales and North Carolina. In addition, the reported number of H274Y cases has exploded in the United States and the Netherlands in recent weeks, and these new isolates will undoubtedly grow the sub-clades with clusters of H274Y.

[b]Thus, the evolution of H274Y in pandemic H1N1 is following the same path as H1N1 in seasonal flu, which involves H274Y jumping to multiple sub-clades via recombination, leading to an emerging sub-clade with H274Y and receptor binding domain changes.[/b]
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Tue Dec 22, 2009 8:37 pm
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Post Re: Recombinomics Commentary


Tamiflu Resistance Continues to Expand in the United States


http://www.recombinomics.com/News/12310 ... US_51.html


December 31, 2009


A total of 50 cases of oseltamivir resistant 2009 influenza A (H1N1) viruses have been identified in the United States since April 2009, including four newly identified cases since last week. In specimens collected since September 1, 2009, 40 cases have been identified in the United States.

The above comments from the latest (week 51) influenza update by the CDC indicates that the detection rate of H274Y in the United States remains high. Only 61 new cases were tested in the past week, yet four had H274Y. The rate has exploded in the past 6 weeks in the US and across the northern hemisphere, raising concerns that H274Y will pair up with receptor binding domain changes at position 225, leading to widespread resistance as was seen in H1N1 seasonal flu.

Pandemic H1N1 rates continue to decline in the US, setting the stage for a new variant in the next several weeks as temperatures drop across the northern hemisphere. Once again there were no cases of seasonal H1N1 or H3N2 detected, and influenza B detection also fell to zero, increasing concerns that the pandemic H1N1 will crowd out influenza A, as well as influenza B, paving the way for the emergence of multiple variants of pandemic H1N1, which is far more dangerous than seasonal flu.

The steady rise in H274Y, coupled with increases in D225G/N increase those concerns.
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Post Re: Recombinomics Commentary
Commentary

Ukraine Fatalities Spike to 727 - One Day Total 29
Recombinomics Commentary 23:55
December 31, 2009


3,753,896 Influenza/ARI

212,349 Hospitalized

727 Dead


The above totals are from the Ukraine Ministry of Health website, indicating the spike in deaths is incrasing. The 727 dead are 29 higher than yesterday's report and brings the total deaths in the past 96 hours to 94. The rate of one death per dour for the past four days is similar to the rate at the beginning of the outbreak in October.

The five deaths in Donetsk again were the highest, but four deaths in Crimea was associated with the curtailing of holiday activities in an attempt to control the spread (see map). The increases in deaths in Ukraine has attracted international attention because sequences from initial cases had receptor binding domain changes at position 225 (D225G and D225N). These results have led to further analysis by other countries, and sequences released from lung samples for patients in three regions of Russia also had D225G, raising concerns that the change was becoming more widespread, which could lead to a catastrophic wave in the upcoming weeks.

The recent sequences from Russia are distinct from each other signaling continued expansion of D225G/N via recombination, which is also happening with the Tamiflu resistance marker H274Y. Patients with both markers have increased concerns that a much more virulent H1N1 could also be resistant to Tamiflu and Peramivir.

These evolving developments continue to increase concerns, WHO proclamations notwithstanding.

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Post Re: Recombinomics Commentary
http://www.recombinomics.com/News/01291 ... k3_PI.html



Spike In US Pneumonia and Influenza Deaths


Dr. Niman
Recombinomics Commentary 20:09
January 29, 2010


The CDC Week 3 influenza report has been released and once again there were no reported cases of seasonal influenza A. 2 cases of influenza B were reported and the vast majority of cases (98%) were pandemic H1N1, indicating seasonal influenza A has been crowded out in the US.

The frequency of influenza detection has risen slightly and is at the highest level since week 50, signaling the end of the fall wave and the beginning of a winter wave.

However, although the detection rate rose slightly, the P&I deaths spiked higher and are now well above the epidemic threshold and as high as it has been since the peak of the 2008/2009 season.

This dramatic jump in deaths raises concerns that the current H1N1 is more virulent and lethal than the H1N1 circulating in the fall. The early appearance of that virus dramatically increased the P&I, which then declined as the H1N1 levels declined. However, the current jump is well ahead of such a corresponding jump in H1N1, which would support a more lethal H1N1.

Recent reports from Tennessee have described a higher frequency of children with H1N1 entering the ICU as well as a higher percentage dying. These results are early, but mimic the increase in case fatality rate in eastern Ukraine in association with the release of sequences from Oct/Nov which had a strong association of D225G/N with fatal cases, who had significant lung damage.

The low number of positives in the US may reflect a sampling problem. Most tested samples have been from hospitalized patients, but the fatal and severe cases frequently are associated with a rapid movement of virus to the lungs and a high false negative rate. A high false negative rate would keep lab confirmed cases artificially low and preclude aggressive antiviral treatment. Similarly the increase in H274Y may also impact treatments involving Tamiflu.

Since most H1N1 fatalities have been young adults, an age distribution on fatal cases would be useful.

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Post Re: Recombinomics Commentary
D225G/N H1N1 Linkage by Recombination in Australia US China

http://www.recombinomics.com/News/12050 ... enial.html



JCVI has released a series of full sequences at Genbank. Although most were from California, three sequences were from Australia, including A/Australia/6/2009. This sequence begins with two polymorphisms that are found in Australian sequences with D225G or D225N. The third polymorphism is Y233H, which is in the Duke death cluster in North Carolina, which has D225G and D225N. The fourth polymorphism is in the China cluster in Zhejian province, which also has D225G. Thus, the Australia HA sequence has polymorphisms which link together three distinct sub-clades with D225G and D225N, providing compelling evidence for movement of these polymorphisms by homologous recombination.

Although each of these sub-clades contains D225G, D225N, or both, prior to the release of the Australian sequence the sub-clades were unlinked, other than the common changes at position 225. The Australian sub-clade had two polymorphisms that were found in Australian sequences, but the sequences with the position 225 diverged, and one series had A/VICTORIA/2125/2009 with D225N and A/SYDNEY/2503/2009 with D225G, while the other branch had SYDNEY/2501/2009 with D225N. The Australian/6 sequence has both of these polymorphism (A208G and T519C) – see list here and here.

The third polymorphism in Australia 6 is T739C, which codes for Y233H. This polymorphism is found in isolates from the cluster of patients at Duke Medical Center who also had H274Y. In this group there were two sequences with D225G and one with D225N (see list here).

The fourth polymorphism is in the cluster of sequences from Zhejian Province in China. All three sequences have D225G (see list here).

Thus, the one sequence in Australian has polymorphisms found in three distinct sub-lades with clusters of sequences with D225G and/or D225N providing a mechanism of recombination for movement of D225G and D225N from one sub-clade to another. Frequent jumping from one genetic background has been described in the above sub-clades as well as the sub-clade found in Ukraine, Russia, and Norway. Interestingly, the CDC just released another sequence (at GISAID) from North Carolina with D225G (A/North Carolina/53/2009) which is in the Ukraine sub-clade.

Thus, these sub-clades with D225G and D225N are becoming increasingly widespread, which facilitates further jumps, as was seen for H274Y in H1N1 seasonal flu and is being repeated in H1N1 pandemic flu.

The use of homologous recombination allows the polymorphisms to jump from one genetic background to the next, facilitating rapid evolution to escape immune responses generated by prior infections or vaccinations.

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