HEALTH 3/6-3/14/10 Bird&Other Flu Weekly Thread:Norway H1N1 D225G/N Refutes WHO Position

Link to last weeks thread:

2/27-3/5/10 Bird, Other Flu Weekly Thread:Widespread Tamiflu Resistance in Japan

http://www.timebomb2000.com/vb/showthread.php?t=355845

Please See:

Confirmed cases of Swine Flu and maps
http://www.timebomb2000.com/vb/showthread.php?t=330689

Comprehensive Flu Thread, Latest reports, States, Countries, Closings.

http://www.timebomb2000.com/vb/showthread.php?t=330395

Useful Links:

Avian Influenza (Bird Flu)
http://www.cdc.gov/flu/avian/gen-info/facts.htm

Johns Hopkins on influenza:
http://www.iom.edu/Object.File/Master/33/450/Rashid Chotani.pdf

National Avian Influenza Surveillance Information:
http://wildlifedisease.nbii.gov/ai/

CDC
http://www.cdc.gov/flu/avian/index.htm

WHO
http://www.who.int/csr/disease/avian_influenza/en/index.html

CIDRAP
http://www.cidrap.umn.edu/

Official U.S. Government Web site
http://www.pandemicflu.gov/

FAO
http://www.fao.org/ag/againfo/subjects/en/health/diseases-cards/special_avian.html

Public Health Agency of Canada
http://www.phac-aspc.gc.ca/influenza/avian_qa_e.html

European Union
http://ec.europa.eu/dgs/health_consumer/dyna/influenza/country_en.htm

The World Bank
http://web.worldbank.org/WBSITE/EXT...68427~piPK:64168435~theSitePK:1793593,00.html

Norway H1N1 D225G/N Publication Refutes WHO Position
​

http://www.recombinomics.com/News/03051001/D225GN_Norway_Refute.html

Here we report the occurrence of an amino acid substitution, aspartic acid to glycine in position 222 (D222G) in the HA1 subunit of the viral haemagglutinin, in clinical specimens from 11 out of 61 cases analysed in Norway with severe outcome. Such mutants were not observed in any of 205 mild cases investigated (Table), thus the frequency of this mutation was significantly higher in severe (including fatal) cases (p<0.001, Fisher’s exact test, two-sided) than in mild cases.

The above comments from a recent report on position 225 (222 in H1 numbering) changes in clinical samples from Norway, flatly refute WHO comments indicating the changes were not significant or not even worth mentioning when found in a death cluster involving transmitting H1N1 at Duke Medical Center.

D225G/N received increased attention in association with an H1N1 outbreak in Ukraine. Young, previously healthy, adults were dying with a high rate with a hemorrhagic component and the rapid destruction of the lungs. There has been earlier evidence of D225G and D225N linked to.fatal cases, including the detection of D225G in 2 of 5 HA sequences from the 1918/1919 pandemic. Moreover, in 2009 there was evidence of D225G and D225N jumping from one genetic background to another. Thus, the detection of these two changes in the fatal cases in Ukraine was predicted.

However, just prior to the release of initial sequences by Mill Hill, the WHO put up a Ukraine update on Nov 17 stating that no significant changes had been found in the sequences from Ukraine patients, but the next day 10 HA sequences were released and D225G was found in all four fatal cases. Five other sequences from milder cases infected with the same sub-clade strongly suggested the linkage to fatal cases was significant.

The sequence data led to the examination of earlier sequences generated in several countries, including Norway. An alert was issued when Norway found 3 sequences with D225G, which were from two fatal cases and one severe case, which was the first severe cases reported in Norway.

The Norway announcement was followed by a Nov 20 briefing note from WHO, again stating a lack of current evidence linking D225G to an increase in severe or fatal cases.

The Nov statement from WHO was followed by a Dec 2 announcement of transmitting Tamiflu resistance (H274) at a medical center in the UK and the US. The US cluster was at Duke Medical center and 3 of the 4 patients died. The presence of D225G and D225N in that cluster was not disclosed, even though the cluster led to the prediction that D225G/N would be involved.

The WHO then issued a Dec 28 preliminary report on D225G, which claimed that D225G was spontaneous and due to copy errors. The evidence supporting this assertion was based on a lack of clusters in time and space, as well as a lack of linkage to a specific sub-clade.

However, that report was followed by the release of sequences from Ukraine with D225G in the same sub-clade. In Ukraine the sequences were collected over a 2-3 week time frame. There were 37 lung samples from fatal cases and 30 had D225G, D225N, or both (11 samples had both).

The Ukraine sequences formed branches that included sequences with receptor binding domains that were wild type, had D225G, or had D225N. This pattern of different sequences on the same branch, or the same change on multiple branches has led to an explanation of spontaneous mutation. However, the presence of D225G and D225N in the same sample, as well as the high frequency of these changes leads to a much higher likelihood that the movement of the changes from background to background is based on recombination.

These combinations are also found in the Duke cluster, where all four patients were on the same ward and had the same series of markers. However, in spite of this clustering in time and space as well as identical genetic backbones, D225G was found in one patient (in two collections) and D225N was in another, once again supporting acquisition by recombination, as well as transmission, with sporadic detection.

The Norway study also found a statistically significant association of D225G and D225N with severe and fatal cases. They also found D225G on multiple genetic backbones, providing additional support by acquisition by recombination.

The Norway data, as well as sequences from Ukraine, Russia, the United States and other countries show clear clustering and decisively refute WHO claims of lack of significance as well as spontaneous appearances due to copy errors.

Jump In H1N1 Low Reactors In United States Raises Concerns
​

http://www.recombinomics.com/News/03051002/H1N1_LR_US_Jump.html

Five viruses (0.3%) tested showed reduced titers with antiserum produced against A/California/07/2009.

The above comment from today’s CDC week 8 report indicates the number of low reactors reported by the CDC has jumped from 2 to 5 in a week that reflected an all time low in H1N1 testing. The per cent positive is on the rise and is at the highest level in 2010, and as high as it has been since mid-December (week 50), so the number of positive samples is up, in spite of the very limited testing.

Increases in low reactors are expected as the H1N1 evolves away from the current immunity as well as the California/7 based vaccine. The first two low reactors reported by the CDC had N159D and a phylogenetic tree by the US Air Force School for Aerospace Medicine had an isolate, A/Texas/732/2009, from week 47 which had N159S (listed as N156S in tree), which may also be a low reactor. In addition, another isolate, A/OCONUS4/4081/2009 from week 5, had N159K but since the isolate was outside of the continental US it is unlikely to be one of the new low reactors. However, as of Feb 2, 472 HA sequences had been sent to the CDC, so additional examples of N159K in the US may be present. Moreover, the detection of three different changes at the same position (N159D, N159S, N159K) suggest that this position may play a significant role in immunological escape in the upcoming wave. This change is adjacent to G158E, which has also been linked to low reactor status in isolates from Germany, as determined by the CDC and Mill Hill. The changes are in addition to K157E, which has been reported as another low reactor.

These recent reports of low reactors, in addition to D225G which has been designated a low reactor by Mill Hill and was recently shown to have a statistically significant association with severe and fatal cases in Norway, and higher frequencies have been reported for Ukraine, raise concerns that current vaccinations and immunity from prior H1N1 infections will have limited efficacy against newly emerging variants of H1N1.

All three pandemics last century had a fall and spring wave. Therefore, a wave is expected to begin in the near term, and several data sets, including this week’s uptick in samples testing positive for H1N1, suggest this third wave may have already begun. Unfortunately, H1N1 surveillance at this time remains weak. Last year the first H1N1 was from a late March collection, even though data from Mexico indicates the pandemic began 1-2 months earlier. The limited testing in the US and other countries worldwide increases the likelihood of delayed detection again. A year ago that delay led to a late start in vaccine production, which delivered product after the fall outbreak had peaked, creating a large surplus.

Although this vaccine has been recommended for the 2010/2011, it is likely to have limited utility, because the low reactors which are being identified now will likely lead to a vaccine target that is not well recognized by an immune response directed at the current target.

The jump in low reactors when surveillance is at an all time low raises concerns that additional changes are not well represented in current reports, and these latest developments are cause for concern.

Roundup: Myanmar experiences bird flu H5N1 re-strike
along with influenza A/H1N1​

http://www.istockanalyst.com/article/viewiStockNews/articleid/3925557

YANGON, Mar. 6, 2010 (Xinhua News Agency) -- Myanmar is experiencing re-strike by bird flu H5N1, detecting more such cases in northwestern Sagaing division this month with some chickens suspected of dying of virulent avian influenza in a poultry farm in Yinmapin township early this week.

It was the third H5N1 case exposed since the avian influenza recurred in Myanmar last month, nearly two years after the country was claimed free from the disease.

The unusual death of chickens were proved to have been caused by highly pathogenic avian Influenza (HPAI) after laboratory tests conducted in Yangon and Mandalay, according to Saturday's official report.

The findings have been further reported to the Food and Agriculture Organization (FAO) and the World Animal Health Organization (OIE).

The authorities have culled chickens and destroyed eggs bred in the farm as its control measures.

According to the Livestock Breeding and Veterinary Department ( LBVD), the first case in the bird flu recurrence this year occurred in Yangon's Mayangong township and the second in Mingaladon township both in the same February.

Dealing with the first case in Mayangong township, unusual deaths of some chickens were found in a farm where 2,500 chickens were bred and the suspected deaths were proved to have been caused by the HPAI after experiment.

As for the second case in Mingaladon township, two domestically- bred chickens died of virulent avian influenza in a poultry farm and were later confirmed with carrying the virus after laboratory test.

The authorities took control measures by culling hundreds of similar chickens, some ducks and eggs, banning sale of chickens and eggs in six bazaars, 7 km around the poultry farm, where bird flu recurred to prevent the spread of the disease.

People have also been constantly urged to step up bio-security measures, change of livestock breeding system, avoidance of illegal import, transport and trading of chickens and its products, and prompt report of suspected bird flu case.

In April 2008, nearly two years before the bird flu struck Myanmar for the second round, the World Animal Health Organization (OIE) declared Myanmar as a bird-flu-free country three months after the country was proved that there was no residual bird flu virus remained over the period since January of that year.

From February 2006 until December 2007, there were numerous outbreaks of the avian influenza in Myanmar covering 25 townships of six states and divisions.

All of the occurrences were blamed for infecting from abroad especially that the virus was carried into the country by migratory birds from the cold regions in the world infecting local birds, according to the LBVD.

Myanmar reported outbreak of the avian influenza in the country for the first round in some poultry farms in Mandalay and Sagaing divisions in early 2006, followed by those in Yangon division in early 2007, in Mon state's Thanbyuzayat and western Bago division' s Letpadan in July and in eastern Bago division's Thanatpin and in Yangon division's Hmawby in October the same year.

Despite the declaration as a bird-flu-free country, the Myanmar livestock authorities continued to call on the country's people to exercise a long-term precaution against the deadly H5N1 bird flu.

Meanwhile, rapid and widespread infection rate of new influenza A/H1N1 in Myanmar, especially since the beginning of this year also, has prompted control and prevention measures in the country with the World Health Organization (WHO) offering to vaccinate over 4 million Myanmar population free of charge.

The A/H1N1 influenza has also struck across Myanmar from Chin state's Tiddim township and Shan state's Kyaukme to Yangon division's six townships of Bahan, Tamway, Dagon Myothit (north and east), Shwepyita, Hlaingtharya and Hmawby in merely two months.

According to a compiled statistical report of local media, a total of 126 people were infected with new influenza A/H1N1 in Myanmar as of Feb. 20 this year since the outbreak of the disease in the country in June last year.

In 2009, 69 people in Myanmar contracted with A/H1N1, while in the first two months of 2010, another 57 were victimized.

However, no flu death cases were reported in the country.

In the wake of the status, the heath department has urged the people to follow the preventive measures outlined against the virus in order to avoid the spread of virus in the public and to take part in the campaign to prevent the disease.

The authorities also advised all private clinics in the country to report or transfer all flu- suspected patients, who returned from abroad, to local state-run hospitals or health departments for increased surveillance.

More Bird Flu cases in Egypt ​

http://bikyamasr.com/?p=9438

CAIRO: The Egyptian Ministry of Health announced five new cases of human H5N1 avian influenza infection.

The first case is a 53-year-old male from Shobra Elkhima district, Qaliobia Governorate. According to the ministry, the man developed symptoms on February 27 and was hospitalized the same day, where he received oseltamivir treatment.

He is currently in a critical condition.

The second case is a one-year-old boy from Banha district of the Qalyubia Governorate. He experienced symptoms on February 22 and was hospitalized the following day, where he also received oseltamivir treatment. He is in a stable condition, the ministry said.

The third case is a 10-year-old male from Meet Ghamr district, Dakaliya Governorate. He developed symptoms on February 10 and was hospitalized on February 14, where he received oseltamivir treatment. He is in a good condition.

The fourth case is a 30-year-old female from Kellin District, Kafr El-Sheik Governorate. She developed symptoms on February 11 and was hospitalized the following day, receiving similar treatment. She is in stable condition.

The fifth case is a 13-year-old male from Kafr El-Sheik District, Kafr El-Sheik Governorate. He developed symptoms on February 10 and was hospitalized on February 14 to receive the treatment. He is in stable condition.

The cases were confirmed by the Egyptian Central Public Health Laboratories, a National Influenza Center of the WHO Global Influenza Surveillance Network (GISN).

Of the 104 laboratory confirmed cases of Avian influenza reported in Egypt, 30 have been fatal since the virus first appeared in the country in early 2005.

CDC On D225G In Egg Isolates Raises H1N1 Vaccine Concerns
​

http://www.recombinomics.com/News/03071001/CDC_D225G_Vaccine.html

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.

a study done by the WHO Collaborating Centres for Reference and Research on Influenza (WHOCC) in Atlanta located in the Centers for Disease Control and Prevention (CDC) found the D222G substitution in 14 virus isolates but not in viruses in the original clinical specimens indicating the D222G substitution in these 14 virus isolates occurred after growth in the laboratory.

The above comments from a response to the recent report from Norway on the significant association between D225G and severe/fatal H1N1 cases, and a WHO preliminary report of D225G, respectively, clearly indicate that the CDC has concluded that samples that yield sequences with D225G in virus grown in chicken embryos are artifact and do not represent D225G in the clinical sample. The CDC has published HA sequences from at least 15 patients with D225G (see list below). Most of these sequences are from initial pandemic H1N1 isolates, collected in April last spring, including the current pandemic vaccine target, A/California/7/2009. These initial isolates were generally from students in California (San Diego County), Texas (San Antonio area), and New York (Queens), and were generally mild. Although D225G was detected (alone or as a mixture with wild type) in virus grown in eggs, it was not detected in direct sequencing or in virus grown in mammalian cells. This pattern has been interpreted by the CDC as a lab generated artifact, although the data would also support an alternative interpretation of a mixture, with D225G representing a minor component.

D225G is a receptor binding domain change that recognizes gal 2,6 and gal 2,3 receptors. Gal 2.3 receptors are on chicken embryo cells in eggs, as well as many cell types in the human lung. Human receptors in the upper respiratory tract as well as mammalian cells used to grow virus in the lab has largely gal 2,6 receptors. Thus, in direct sequencing of the clinical sample, the major strain will be detected, which would be wild type in samples with D225G as a minor component. The same result would be found in virus grown in mammalian cells because wild type would maintain its numerical advantage. However, virus grown in eggs would have D225G, because the gal 2,3 specificity would allow D225G to out compete wild type, changing the ratio and allowing for D225G detection either alone or as a mixture with wild type, which was found in the April isolates listed below. After April, CDC and most labs used mammalian cells to grow virus in the lab, selecting against D225G and allowing detection only in samples with high levels of D225G.

These ratios would explain why the April cases with D225G were mild. The D225G was at low levels and consequently not detected in direct sequencing or virus grown in mammalian cells, but detected in virus grown in eggs. More recent isolates, including those in Norway, which involved detection of D225G in mammalian cells, but frequently as mixtures with wild type, or via direct sequencing of autopsy lung samples from Ukraine, where D225G, D225N, or both were detected because D225G/N was at high levels, which strongly correlated with fatal cases.

The conclusion that the D225G in the April samples, including A/California/7/2009, may have influenced the decision to select X-181A, which lacks D225G, as the killed vaccine. target. This absence is likely linked to the low yields that led to vaccine shipment delays resulting in the delivery of most vaccine after the fall peak in cases. In contrast, the live attenuated vaccine made by MedImmune had D225G and grew well in eggs, leading to shipment weeks ahead of the killed vaccine.

Thus, the CDC conclusion that D225G identified in eggs was a lab artifact may have had significant consequences regarding the selection of the vaccine target in April, as well as the recent recommendation that the vaccine can be used in the 2010/2011 season, when D225G may be much more prevalent and killing many more people.

D225G (CDC generated sequences)

name age/gender date
A/Texas/05/2009 16M 4/15/2009
A/Texas/11/2009 9M 4/23/2009
A/Georgia/01/2009 12F 4/27/2009
A/New York/04/2009 17F 4/2009
A/Illinois/10/2009 55M# 7/31/2009
A/North Carolina/53/2009 53M 10/18/2009

mixture with wild type
A/New York/31/2009 14F 4/24/2009
A/New York/11/2009 16F 4/25/2009
A/California/13/2009 4/21/2009
A/Texas/10/2009 6F 4/23/2009
A/California/07/2009 54M 4/9/2009
A/Nebraska/02/2009 48M 4/2009
A/Texas/04/2009 16M 4/14/2009
A/Utah/42/2009 28F$* 7/24/2009
A/North Carolina/39/2009#* 43F 10/15/2009

# = H274Y
* = known or likely fatal
$ = D225N mixture

CDC Reliance on Negative D225G/N Data Raises H1N1 Concerns
​

http://www.recombinomics.com/News/03071002/CDC_D225G_Neg.html

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 infected with the Tamiflu resistant 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.

CDC In Denial On H1N1 D225G/N Significance
​

http://www.recombinomics.com/News/03071003/CDC_D225G_Denial.html

However, Dr. Nancy Cox, director of the Influenza Division at the US Centers for Disease Control and Prevention (CDC), said global H1N1 data so far do not show a clear association between the mutation and severe illness.

The above comments are by an agency in denial. The association between D225G and D225N with severe and fatal H1N1 infections is overwhelming. The recent report from Norway indicated that 9/27 (33%) of fatal cases in Norway had D225G or D225N. However, in Ukraine 30/37 (81%) of autopsy lung samples have D225G and/or D225N (11 have both). Similarly, D225G was in 40% of the fatal lung samples from 1918/1919. The data from Ukraine and 1918 are from direct sequencing.

The CDC has tried to limit the number of positives by excluding samples that are positive in culture but negative in the original sample. However, there is little evidence to support CDC’s position that the negative data is true, while the positive data is false. Direct sequencing lacks sensitivity, and both D225G and D225N are frequently detected as mixtures, either with wild type or each other. The failure to find such mixtures is not unexpected, especially if one polymorphism (D225G) is at low levels, as would be expected in mild cases. The low level of D225G would provide a mechanism for transmission, instead of the WHO position that D225G is not transmitted and spontaneously appears again and again.

The Ukraine data does not support repeated spontaneous mutations. D225G and D225N are found in 81% of fatal cases that occurred over a short time frame. A sudden increase in spontaneous errors at the same time at two positions has no scientific basis. The proposals are little more than ad hoc arguments that are not supported by the data.

Similarly, the sudden appearance of the same change on multiple genetic backgrounds is not supported by a “random mutation” model, which is WHO’s working hypothesis. These models are destroyed by the actual data, which is why the significance of D225G and D225N are downplayed and putative lab artifacts are introduced in attempts to discount and discredit the actual data, even when generated by the CDC itself.

The random mutation model is further discounted by the presence of two changes linked to the same codon, as well as the widespread detection of another polymorphism, D225E, at the same codon, which is associated with milder disease in several countries in western Europe, including UK, Spain, and Italy, where frequencies from 20-50% have been reported.

The RBD changes are clearly transmitted and clustered in time and space. The lack of data supporting random mutations, and the clustering of data which clearly refutes the model, have created an agency in denial of the overwhelming data linking D225G and D225N to severe and fatal cases, as well as the movement of these changes from one genetic background to another via recombination.

H1N1 G158E Low Reactors In Japan Raise Vaccine Concerns​

http://www.recombinomics.com/News/03091001/H1N1_G158E_Japan.html

NIID released a small series of sequences at GISAID, including two from Japan. Both had G158E, the polymorphism found in two isolates from Germany, which were designated low reactors by the CDC and Mill Hill. A/YAMAGATA/721/2009 was collected Dec 8 and had a mixed signal at position G158E. The other was A/IWATE/1093/2009 and collected Oct 27.

The presence of G158E in both sequences from Japan raises concerns that low reactors are becoming more prevalent. In the two isolates from Germany, the only non-synonymous change is G158E. This change has also been found in two isolates with D225G, one in Italy and one in Russia. Mill Hill has also designated D225G as a low reactor. Moreover, the Yamagata isolate also has D225N, which like D225G has been linked to severe and fatal H1N1 cases.

The release of the sequences from Japan follows the CDC week 8 report on H1N1 in the United States, which increased the number of low reactors reported by the CDC in the US from 2 to 5. The two earlier reactors had changes at the adjacent position, N159D. Moreover, sequences released from the Air Force had N159K and N159S. The three changes at position 159 (N159D, N159K, N159S) as well as G158E all map to the same antigenic site, which is distinct from D225G or D225N, raising concerns of multiple low reactor polymorphisms aggregating via recombination to produce additive effects leading to widespread vaccine failure.

H1N1 Low Reactor G158E D225N Recombinant In Japan​

http://www.recombinomics.com/News/03091002/D225N_G158E_Japan.html

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. D225G, has affinity for gal 2,3 receptors found in chicken embryos in eggs, and therefore 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, 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 D225G confers low reactor status, and can recombine with another low reactor polymorphism, like G158E.

These recent results raise serious question about H1N1 surveillance represented by public sequences, and continue to be hazardous to the world’s health.

In Brief: New plan to fight bird flu in Egypt ​

http://www.irinnews.org/report.aspx?ReportID=88350

CAIRO, 8 March 2010 (IRIN) - Egypt is moving to curb the spread of avian influenza (H5N1) after a recent upsurge in infections, the Egyptian Health Ministry says.

The sale of poultry between any of Egypt’s 29 governorates is to be banned, and a major Health Ministry-led awareness campaign will alert the public to the dangers of raising birds at home, Sabir Galal, deputy chief of the Veterinary Medicine Section at the Health Ministry, told IRIN. “Bird flu has become endemic in this country… The fear now is that the virus can assume more dangerous forms in the days to come,” he said.

The Ministry also said it would stop inoculating birds after vaccines had proved incapable of stopping the virus from spreading.

With 105 infections to date, including 30 deaths, Egypt is the world’s third most affected country by avian influenza, according to the World Health Organization.

H1N1 Low Reactor G158E In Athens Greece
​

http://www.recombinomics.com/News/03101002/G158E_Greece.html

Recently released sequences from Mill Hill at GISAID included a sequence, A/Athens/16606/2009, from Greece collected on December 26, 2009. The sequence contained G158E, which was found in low reactors designated by Mill Hill and the CDC. Although only a partial sequence was released, it contained E377K, which was also in the Hyogo/1597/2009 isolate from Japan, indicating G158E was transmitting. Similarly, four of the G158E sequences from Japan also had A200T including one sample with H274Y. Another sample in Japan had D225N, while samples in Italy and Russia had D225G.

These various combinations signal movement of G158E from genetic background via recombination, which was also seen for D225G, D225N, and H274Y. D225G has also been designated a low reactor marker, leading to concerns that these markers will be more common in upcoming waves, including the wave which may be beginning now.

Increases in these markers would lead to more severe and fatal cases as indicated by the association of D2225G/N in Ukraine, Norway, and Greece. Although current approaches has yielded high frequencies of D225G, the detection of D225G in egg isolates from mild cases raises concerns that low levels of D225G may be widespread, and increases may be missed with current assays which focus on direct sequencing and growth of virus in mammalian cells, which failed to identify D225G in milder cases, which had D225G in egg isolates. However, this failure may be linked to mixtures in these samples, which may be less common as immune responses reduce the level of wild type receptor binding domains.

H1N1 Tamiflu Resistance and D225G In 2010 Greek Case​

http://www.recombinomics.com/News/03101001/H274Y_D225G_Greece.html

Recently released HA sequences from the Aristotle University of Thessaloniki at Genbank in a report entitled “Molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza H1N1 2009 viruses associated with severe and fatal infections” includes 9 sequences from 2010 and 3 have D225G. One (8F) of the three, A/Thessaloniki/225/2010, was also Tamiflu resistant (H274Y). Although the clinical status of each patient was not included, the title of the submission suggests that these samples were from severe and fatal cases.

In addition to the three cases from 2010, three additional cases, had D225G in samples collected in mid-November, again pointing toward higher frequencies of D225G in more recent cases. Late October through mid November collections form autopsy lung in Ukraine had 27/37 samples positive for D225G. D225N, or both. The sequences from Greece were from upper respiratory tract collections. D225G in lung samples may have been detected at a higher frequency.

In Norway, samples with D225G were exclusively found in severe (3) or fatal (8) patients, which were statistically significant.

The three samples positive for D225G in 2010 represents the highest number reported from one country in 2010, which includes the first report in 2010 of D225G and H274Y in the same patients. Last year D225G with H274Y was reported in patients in France and the United States, including the Duke death cluster.

Specific patient outcomes and testing of samples from the lower respiratory tract would be useful.

Low-Path Bird Flu Detected in Denmark​

http://www.thepoultrysite.com/poultrynews/19698/lowpath-bird-flu-detected-in-denmark

DENMARK - Low-pathogenic avian influenza (LPAI) has been found on a duck farm during routine surveillance.

The veterinary authority sent an Immediate Notification dated 8 March to the World Organisation for Animal Health (OIE).

The report says that the virus was found on a farm at Fuglebjerg on the island of East on 5 March. The affected population was a breeding farm with 190 mallards and five hens. The suspicion arose on 5 March 2010 due to positive samples taken in connection with the surveillance programme for avian influenza. All the birds have been destroyed.

The virus appears to be an H7 sub-type but serology results are pending.

The last outbreak of avian flu in Denmark was in May 2008.

H1N1 Greek and Russian D225G Sequences Identical​

http://www.recombinomics.com/News/03101003/D225G_Greece_Russia.html

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.

Low Reactor G158E In 2010 Swine Pandemic H1N1
​

http://www.recombinomics.com/News/03111001/G158E_Swine_MN.html

Recently released sequences by the US National Veterinary Labs in Ames, IA included two sequences from MN swine, A/swine/MN/8762-1/2010 and A/swine/MN/8762-2/2010, which have G158E.

Both sequences have a collection date of February 26, 2010, which represents the most current public 2010 pandemic H1N1 sequence and the first examples of G158E in swine pandemci H1N1. This polymorphism has been linked to a “low reactor” designation by Mill Hill and the CDC. It is also in the antigenic site associated with immunological escape from neutralizing monoclonal antibodies which react with 1918 and 2009 pandemic H1N1. The number of reported sequences with G158E is on the rise in Asia and Europe. Examples have also been reported in the United States although CDC has reported these as examples as California/7 like, raising questions about recent antigenic characterizations. WHO has acknowledge inter-lab differences and has proposed a new standard using pooled human antibodies, which may produce uniform test results with lowered sensitivity.

The detection of G158E in February swine H1N1 isolates provides additional signals that the rate of H1N1 evolution is increasing. These sequences have D1381G, which is widely detected in human sequences including those in Ukraine and Norwat with D225G (see list here). However, the Minnesota swine isolates have a number of non-synonymous changes which are largely limited to swine isolates See list here here here here). These may be present in human 2010 sequences, but release of such sequences is significantly delayed, and only a handful of human 2010 sequences have been released. The largest number were just released by Greece, and 3/9 had D225G, which is another polymorphism being reported at increasing frequencies.

The latest sequence from declineChina, A/Beijing/22811/2009, was also just released. The December, 2009 isolate has 9 recently acquired non-synonymous changes in HA, once again signaling increased H1N1 evolution.

As H1N1 evolution is increasing, antigenic characterization tests are increasingly suspect. Similarly, the number of samples being collected and the release of data is on a , while the start of a new wave is being signal.

The current degrading H1N1 surveillance is becoming increasingly hazardous to the world’s health.

Bird flu still rampant in Jambi Province​

http://www.antara.co.id/en/news/1268381869/bird-flu-still-rampant-in-jambi-province

Jambi (ANTARA News) - Bird flu had remained a serious threat in Jambi province because 2,000 chickens had died over the past month, a local health official said.

Almost all districts and towns in Jambi had practically been infected with the deadly virus, Head of the province`s animal husbandry office, Hanis Lubis, said here Friday.

"Based on the reports that I have received two thousand chickens have suddenly died over the past month. This number represents a 50-percent increase compared with last year`s figure," she said.

The wider spread of the Avian Influenza in Jambi this year was mainly caused by the longer rainy season than last year, Lubis said.

Apart from the weather factor, the destructive habit of chicken breeders also contributed to the wider spread of the deadly viruses.

Lubis said local chicken breeders remained accustomed to throwing the dead chickens into rivers.

"Instead of throwing them away, the chickens must be burned before burying them to halt the bird flu virus spread," she said.

The dead chickens mostly belonged to local residents but there were no reports of Avian Influenza virus transmission to human.

Despite the absence of the human case of bird flu infection, local residents and chicken breeders need to be cautious with the unwanted eventualities, she said.

Veterinarians had been deployed to the affected regencies and towns to intensify public awareness campaigns.

"We urge locals to immediately inform us if they find a big number of suddenly-dead chickens. They must also avoid a direct contact with those chickens," she said.

The bird flu attacks remain serious problem for almost all provinces in Indonesia particularly during rainy season.

The bird flu viruses attacking the country is classified as "highly pathogenic avian influenza".

According to the World Health Organization (WHO), avian influenza or "bird flu" is a contagious disease of animals caused by viruses that normally infect only birds and, less commonly, pigs.

The WHO has warned that the infection with avian influenza viruses could spread very rapidly through poultry flocks.

Indonesia has 33 provinces. Only three have been confirmed free from the threat of bird flu viruses.

Indonesia has been dealing with bird flu since 2005. However, the H5N1 type influenza is also known to have attacked chicken and birds in other Asian countries, such as Thailand, Cambodia, China, and Vietnam.

WHO Flu Research Lab Planned for Indonesia​

http://www.thejakartaglobe.com/news/who-flu-research-lab-planned-for-indonesia/363427

The World Health Organization has asked Indonesia for permission to build a bird flu research laboratory in the country, fulfilling a longstanding desire by domestic health care officials to build such a collaborative center, the health minister said on Wednesday.

“WHO has requested that Indonesia become the location of a flu research laboratory, better known as a World Health Organization Collaborative Center, and I have said that we agree,” Health Minister Endang Rahayu Sedyaningsih told the conference titled “One World, One Health: Winning the Battle Against the Diseases of the 21st Century” in Jakarta.

Endang did not provide details. “Everything is still in process,” she said.

Although the country’s bird flu management has been relatively successful, Endang said the danger still existed, noting that the virus continued to be found.

“There has been one human bird flu case found so far this year even while the effects of the H1N1 influenza [swine flu] pandemic are still being felt around the world,” she said.

Endang recently told a news conference that there were four WHOCCs located around the world — in Atlanta, Tokyo, Melbourne and London. She said all WHOCCs until now were built in developed countries even though the disease was a world-wide issue and especially threatening to developing countries.

Endang said that a few years ago Indonesia would send its virus samples to an overseas WHOCC but had difficulty accessing the test results, which pointed to the need for the country to have its own laboratory.

Deputy Agriculture Minister Bayu Krisnamurthi said Indonesia had been fighting for the right to build a WHOCC for some time, especially given that it had the world’s highest number of bird flu fatalities.

Widespread H1N1 Low Reactor G158E In US Raises Concerns
​

http://www.recombinomics.com/News/03121001/G158E_US.html

Recently released sequences by the CDC at GISAID have G158E, raising concerns that these low reactor sequences are becoming more widespread. G158E is in earlier isolates from Germany, A/Bayern/62/2009 and A/Bayern/69/2009. Although these two isolates have synonymous changes that distinguish the two viruses from each other, the only recent non-synonymous change was G158E. Mill Hill ran an antigenic Characterization test on both, and declared both “non-reactors”.

The CDC also tested Bayern/69 and also declared it a low reactor. However, the recent (October and November) isolates in the US were tested, but not labeled low reactor. Included in the recent sequences with G158E are three isolates from Washington (A/Washington/60/2009, A/Washington/61/2009/ and A/Washington/65/2009). Although all three isolates have G158E, they have different combinations of other polymorphisms, placing them in different sub-clades, signaling recombination..

The most recent isolate, Washington/65 also has E377G. Several other US isolates with G158E have E377K (Texas/65/2009, Idaho/10/2009, Minnesota/18/2009, Arizona/18/2009, Hawaii/29/2009) as well as isolates in Japan (A/Hyogo/1597/2009) and Greece (A/Athens/16606/2009), indicating this sub-clade has spread significantly.

The reason behind the failure of the CDC to classify some of these recent isolates as “low reactors” is unclear. Prior to last week, only two low reactors were reported by the CDC in the US, and both had a change at the adjacent position N159D. Both position map to the same antigenic site and a recent paper on the effectiveness of 1918 anti-sera on the 2009 H1N1 noted that escape mutants also mapped to this antigenic site. However, a recent WHO reported noted that there were differences between labs running antigen characterization tests, and a new universal reference anti-sera had been created using pooled sera from patients, which was available in late 2009. This new reagent may have produce a negative low reactor result because a pooled sera would have a wider range of antibodies, relative to the traditional tests, which use ferret antibodies from experimental animals immunized with the vaccine target.

Since there are approximately 5 amino acid differences between California/7/2009 and the consensus H1N1 sequence, the ferret reference sera may be more sensitive than pooled sera, especially if the sera is from patients infected with H1N1 instead of being vaccinated with California/7. If the sera is from vaccinated patients it is unclear which vaccine was used because there are at least three different sequences in various commercial products. Even within the US the killed vaccine has Q226R, while the live vaccine has D225G. In Europe the adjuvented vaccine has both, but as mixtures with wild type. These differences could affect results, especially for D225G, which Mill Hill designated as a low reactor. In a recent antigenic characterization of an isolate from Ukraine, which had G158E and D225G, the CDC did not classify it as a low reactor, suggesting that the assay being used by the CDC lacks sensitivity.

These differences involving polymorphisms that had been independent mapped to antigenic sites, and have tested as low reactors in earlier assays raise serious concerns about the validity of the recent negative results by the CDC, especially if pooled anti-sera is being used.

Uniform false negatives remain hazardous to the world’s health.

Ha Noi woman tests positive for bird flu ​

http://vietnamnews.vnagency.com.vn/...a-Noi-woman-tests-positive-for-bird-flu-.html

HA NOI – A 25-year-old woman from Soc Son District, Ha Noi was tested positive for avian influenza virus type H5N1 or bird flu on Thursday, according to the Preventive Medicine and Environment at the Ministry of Health.

The patient, who is now in intensive care in Bach Mai General Hospital, caught the virus on March 5, allegedly from ill and dead poultry that were near her home.

Bird flu hits Thakurgaon poultry farm ​

http://www.thedailystar.net/newDesign/news-details.php?nid=129868

It was avian influenza commonly known as bird flu that triggered death of chickens at a poultry house in Thakurgaon district headquarters in the last couple of days, said livestock officials yesterday.

District Livestock Officer Mosaddekur Rahman told The Daily Star that a total of 395 parent chickens at Kazi Breeders Limited Yakubpur branch had died until Thursday noon starting from Wednesday night.

As the mortality rate was abnormal, the farm authorities informed the livestock office, he said.

Livestock officials immediately collected samples and sent those to Joypurhat Field Decease Investigation Laboratory Thursday evening. Samples were also sent to Bangladesh Livestock Research Institute (BLRI), Savar.

Meanwhile, fearing bird flu infection, the farm authorities isolated the parent chickens and eggs and imposed restriction on bringing the birds and eggs to other side of the poultry.

Yesterday around 5:45pm the BLRI sent a fax massage to the District Livestock office confirming the bird flu detection.

A 50-member team from District Livestock Office with the help of district administration started a culling drive yesterday around 7:30pm.

About 1,17,626 parent stock and about 2,10,000 eggs would be destroyed there, said the district livestock officer.

Thakurgaon Deputy Commissioner Munshi Shahabuddin Ahamed told the Daily Star that they have put a ban on carrying, purchasing and selling of chicken, duck, pigeon, eggs and all sorts of birds from the farm.

DC Shahabuddin said we would also try to make people aware of the influenza.

The district administration placed a three-month ban on trading of such birds in the area.

Spike In H1N1 In Texas and Alabama Raises Concerns​

http://www.recombinomics.com/News/03141001/H1N1_TX_AL.html

Doctors have noticed a small rise in the number of flu cases at Texas Children's Hospital in the past few weeks, and health officials are hoping the coming months won't bring a repeat of last spring's rash of swine flu illness.

There were two culture-confirmed cases of Type A influenza in December, three in January and seven in February — all found to be of the swine flu subtype, Demmler-Harrison said.

Five cases of Type A influenza surfaced in the first week of March, but tests are still pending to determine if those are swine flu, she said.

At Andalusia Regional Hospital, positive flu cases are averaging about one per day, said Candie Northey ARH’s director of infection.

“We can’t confirm whether these are H1N1 or typical seasonal flu because the tests we utilize are simple ‘quick tests’ and only reveal positive or negative flu,” Northey said. “We have noted five positive tests through our lab in the last five days, and while this is not a lot, I do feel the need to refresh everyone on how to prevent the spread of flu.”

The same can be said at Opp’s Mizell Memorial Hospital, where Marsha Seppala, MMH’s director of case management and infection prevention, said the first of the year started out relatively slow for positive flu cases.

The above comments describe increases in Type A influenza cases in early March in the Houston area, as well as two cities in southern Alabama. In the US, seasonal type A flu cases have virtually disappeared. In 2010 there has only been one confirmed seasonal H1N1 in the entire country, and there has also only been one H3N2 case in the past five weeks. Therefore, the increase in type A described above is an increase in pandemic H1N1.

This increase is not unexpected. Although reporters have been requesting a declaration that the 2009/2010 pandemic has ended, each of the past three pandemics had a fall and spring wave. Moreover, the seasonal flu was crowded out, as has happened with seasonal H1N1 and H3N2. Therefore, there is no evidence against a spring wave.

In the US the first confirmed swine flu cases were on samples collected March 30 and April1, 2009. However, the confirmed cases were from clusters involving earlier infections, which would have involved swine H1N1 infections that began almost exactly one year ago. That outbreak peaked in May, and a similar time frame is expected for 2010.

Pandemic waves begin when a high percentage of the target population has little or no immunity and end when the population does have immunity. A new wave is driven by a virus that has evolve away from the immunity, as indicated by “low reactor” status, such as those with G158E. This polymorphism began to expand in the US during the fall wave, and then began to appear in larger numbers in Japan in December.

However, the appearance of G158E in the most recent public sequences, two Feb 26 isolates from swine in Minnesota is cause for concerns. These two isolates represent the same sub-clade found in Ukraine, Russia and Norway that had D225G and were linked to fatal cases. However, the virus is circulating in swine and isolates in Illinois and Minnesota have picked up the same swine polymorphisms. The detection of the same H1N1 in Minnesota and Illinois indicates the virus is circulating in swine, and the two most recent isolates from Minnesota have added G158E. Since these isolates are the pandemic H1N1 sub-clade that is widespread in Ukraine, it can jump back into the human population and introduce the swine acquisitions. Both G158E and D225G are common in swine. Moreover, 1918 sequence have an equal mixture of human and swine H1N1polymorphisms acquired via recombination, which would also produce new acquisitions in the Minnesota swine, including G158E.

Thus, the above reports signal the start of a spring wave, and the increases in low reactor polymorphisms like G158E and D225G are likely candidates for increases due to positive selection pressure, which would lead to more severe and fatal cases.

Sequence data on the emerging cass in March would be useful.

Bangladesh slaughters 117,000 birds over avian flu​

http://www.google.com/hostednews/afp/article/ALeqM5g26pBhkelbG9hEci80YTQTErZyOw

DHAKA — At least 117,000 chickens were destroyed in northern Bangladesh Sunday after avian flu outbreak on one of the country's largest poultry farms, a local official said.

The deadly H5N1 strain of flu was detected on Saturday when 400 chickens died suddenly at the Kazi Farms complex in Thakurgaon town, district livestock chief Mosaddekur Rahman told AFP.

"Tests confirmed presence of the H5N1 bird flu in 15 sheds of the farm and we ordered destruction of all 117,600 layer chickens," he said.

Kazi Farms is Bangladesh's largest poultry bird and egg producer. General manager of the company Ataur Rahman said another 200,000 eggs had also been destroyed in the single largest outbreak of bird flu in the country.

"Our loss will be more than 400 million taka (six million dollars)," he told AFP.

Bangladesh was hit by bird flu in February 2007, when more than one million birds were slaughtered on thousands of farms across the country.

The last major outbreak was in November 2008 when 10,000 birds were culled over a two-month period, with smaller outbreaks detected in 2009.

Bangladesh's poultry industry is one of the world's largest, producing 220 million chickens and 37 million ducks annually.

The country reported its first confirmed human case of bird flu in May 2008, but the government said the 16-month-old baby who contracted the virus recovered.

Mill Hill ran an antigenic Characterization test on both, and declared both “non-reactors”.

Click to expand...

Niman may want to correct the "non-reactors" statement. Both are low reactors.

Link to next weeks thread:

3/15-3/22/10 Bird&Other Flu Weekly Thread:Potential Pandemic H1N1 Vaccine Mismatches

http://www.timebomb2000.com/vb/showthread.php?t=356908