RECOMBINOMICS

[dothedd]

S246N and H274Y Tamiflu Resistance Clinical Concerns
Recombinomics Commentary 16:00
June 13, 2011


Pharmacokinetic data would suggest that the maximum drug levels achieved via the recommended dose easily exceed the observed IC50 values of the S247N mutant [11], and therefore the variant is unlikely to be clinically resistant.

The above comments in the recent report on the rapid clonal expansion of S246N (S247N in N1 numbering) is widely disseminated in media reports and refers Tamiflu data from a 1999 publication. However, clinical data data on actual patients treated with Tamiflu raises serious concerns about the effects of a six fold reduction in the effective dose of Tamiflu (or a corresponding 3 fold reduction in the effectiveness of Relenza).

Most of the clinical data has been on patients treated with Tamiflu, since it has been approved for a longer time period and has been used in the treatment of prevention of H5N1 as well as season and pandemic influenza.

Tamiflu is most frequently used in Japan, and earlier use of lower doses in children led to widespread resistance in seasonal H3N2 linked to genetic changes at multiple positions. Earlier in vitro studies using all nine neuraminidase serotypes indicated Tamiflu was less effective against N1, which would help explain clinical issues with H5N1, seasonal H1N1, or pandemic H1N1 associated with H274Y (H275Y in N1 numbering).

Resistance in H5N1 was initially associated with the appearance of H274Y and S296N in a contact treated prophylactically with Oseltamivir (and prophylactic treatment is used at 50% the treatment dose). This two-fold difference led to the use of Tamiflu at double the recommended dosage as well as clinical trials using Tamiflu at a double dose for H5N1.

However, data on Tamiflu treatment for seasonal and pandemic H1N1 is more extensive and clinical results again raise concerns that H1N1 (pandemic and seasonal) can effectively evade the effects of Tamiflu). For seasonal H1N1, H274Y began to appear in patients not receiving Tamiflu signaling fitness of H1N1 with H274Y. Low levels were seen for clade 1 (New Caledonia), and clade 2C (Hong Kong), but dramatic increases were seen in clade 2B (Brisbane/59) in the 2007/2008 season. High levels (above 50% were reported in northern Europe) and then the level of H274Y rose to 100% in the southern hemisphere in 2008, followed by 100% of H1N1 in the northern hemisphere in the 2008/2009.

The 2009 H1N1 pandemic displaced / replaced seasonal H1N1, but H274Y continued to be a concern. Initial cases again involved H1N1 treated prophylactically, again signaling concerns that a two difference in dosing could lead to infections. Similarly H274Y was identified in patients who had not been treated with Tamiflu, and increasing community transmission has been noted, most recently in Delaware and Maryland.

The spread of S246N, which reduces the efficacy of Tamiflu six fold, would likely accelerated the spread of H274Y, which lowers the efficacy of Tamiflu 600 fold and is additive with S246N.

Moreover, H274Y is circulating as a mixture (and levelss below 50% are not being reported), leading to rapid detection after the start of treatments as seen in the H1N1 fatal infection (40M) in Perth. The sample collected prior to treatment, A/Perth/30/2011, had S246N, while sample collected 5 days after the start of treatment, A/Perth/29/2011, has S246N and H274Y.

Thus, the 6 or 3 fold reductions in effectiveness for Tamiflu and Relenza respectively cased by S246N, can have profound and far reaching clinical effects, which cause considerable concern due to the rapid spread of S246N in Singapore and Australia.

[dothedd]

Widespread S246N Tamiflu Resistance Clusters Raise Concerns
Recombinomics Commentary 20:15
June 13, 2011

The recent report on “moderate” Tamiflu and Relenza resistance (6 and 3 fold reductions in efficiencies) linked to S246N has raised concerns that this genetic change will become widespread and the modest reduction in efficiencies will lead to an increase in changes that have more severe consequences, such as H274Y, which reduces Tamiflu effectiveness by 600 fold, and syngergizes with S246N to generate 6000 fold reductions.

There are concerns that similar synergies will be seen in isolates with S246N and changes at position 222 (I222R or I222K) which reduce Relena effectiveness rendering the two major neuraminidase inhibitors useless.

The paper noted the increasing levels of S246N in Singapore and Australia due to rapid clonal expansion. However, the paper included a phyogentic tree showing S246N on multiple H1N1 backgrounds, signaling expansion via recombination, which appended S246N onto multiple gentic backgrounds.

However, a close examination of these additional cases, most of which were from 2009 or 2010, also formed small clusters, signaling additional clonal expansion and an absence of a selection penalty. Two identical sequences with S246N (A/Jiangxi-Donghu/SWL131/2010 and A/Jiangxi-Donghu/SWL157/2010) were collected a week apart from the same province in China. Similarly three other sequences which matched each other (A/Athens/INS342/2009, CY062875 A/Athens/INS161/2009, A/Athens/INS350/2009) were collected days apart from three patients in Athens). In other clusters the sequences were closely related to each other including three isolates from the United States (A/North Carolina/03/2010, A/North Carolina/05/2010, A/Minnesota/02/2010) or two isolates from eastern Europe (A/Croatia/18576-1/2010 and A/Slovenia/234/2011). Thus, each cluster represents an independent introduction of S246N via recombination), and the clustering in time and space presents clonal expansion of each introduction. These clusters are smaller than those reported for Singapore and Australia in part because in 2009 and 2010 there was little immunity in those under the age of 60 so novel sub-clades did not dominate.

In the 2010/2011 new and larger sub-clades began to appear such as the S188T and S186P sub-clade that were large and widespread. Another sub-clade with changes near the larger receptor binding domain, which had R208K and I219V in addition to D100N and V252L began to expand in Sweden, Iraq, and Iran in late 2010, early 2011. More evolved versions of this sub-clade were seen in large numbers in Italy (Milan and Pavia) and these sequences were very closely related to the sequences in Singapore and Australia. Most of the sequences from Italy did not include NA, so the presence or absence of S246N could not be determined for most public isolates, but S246N was not in the few NA sequences published. However, the large number of sequences from this sub-clade indicated it could spread rapidly, which happened in Singapore and Australia after S246N was acquired.

Thus, the prior history of small clusters in 2009 and 2010 on multiple genetic backgrounds, coupled with the rapid spread in Australia and Singapore after S246N was acquired raises concerns that S246N will be common in the current flu season beginning in the southern hemisphere, and this change will lead to significant clinical consequences involving additional resistance changes including H274Y and I222R/K.

[dothedd]

Pneumonia and Influenza Death Toll In El Paso Increases To 116
Recombinomics Commentary 23:15
June 15, 2011

The week 22 Pneumonia and Influenza (P&I) deaths for El Paso, Texas set another weekly record. The 10 deaths raised the total for the past 9 weeks to 116. In the past 9 weeks, record levels were reported for weeks 14, 15, 18, 19, 20, 21 and 22. Thus, even though week 22 is two weeks after the official end of the 2010/2011 flu season for the northern hemisphere, deaths in El Paso remain at record double digit levels. The P&I death rate for these nine weeks continues at a rate that is above 12%, which is more than 1.5 times the national epidemic threshold for the period.

The spike in deaths began in week 14, when the 19 reported deaths were 1 death shy of the weekly record reported for El Paso in the past 15 years at the CDC website, which maintains the weekly data fro the top 122 cities in the US over the past 15 years. The record of 20 was broken in week 15, when 24 deaths were reported. The numbers declined in weeks 16 and 17, but a new weekly record has been set for each of the past 5 weeks, even though lab confirmed cases have steadily declined to baseline levels as the flu season ended.

The record number of P&I deaths in El Paso has not been addressed by local or national agencies and these high levels are not reflected in lab confirmed cases raising concerns that the sampling and associated testing grossly under estimates the number of influenza deaths, particularly for H1N1. In the past 9 weeks the number of deaths in the 25-64 age group has been over represented, providing additional data linking the spike in deaths to unreported / undetected H1N1 infections, consistent with the linkage between the start of the increases and the outbreak of Chihuahua H1N1 across the border in Juarez, Mexico.

This outbreak lead to a pandemic alert, and the above data suggests that the cases are grossly under represented by the lab confirmed numbers. The pandemic alert was linked to the detection of the D225N receptor binding domain (RBD) change in the upper respiratory tract of severe and fatal cases, but sequences demonstrating a high frequency of D225N in Chihuahua have been withheld.

The withholding of these sequences, and the lack of comment on the record number of deaths in El Paso, continue to raise pandemic concerns.

[dothedd]

Pneumonia and Influenza Death Toll In El Paso Increases To 127
Recombinomics Commentary 19:00
June 16, 2011

The week 23 Pneumonia and Influenza (P&I) deaths for El Paso, Texas set another weekly record. The 11 deaths raised the total for the past 10 weeks to 127. In the past 10 weeks, record levels were reported for weeks 14, 15, 18, 19, 20, 21, 22 and 23. Thus, even though week 23 is three weeks after the official end of the 2010/2011 flu season for the northern hemisphere, deaths in El Paso remain at record double digit levels. The P&I death rate for these ten weeks continues at a rate that is above 12%, which is more than 1.5 times the national epidemic threshold for the period.

The spike in deaths began in week 14, when the 19 reported deaths were 1 death shy of the weekly record reported for El Paso in the past 15 years at the CDC website, which maintains the weekly data fro the top 122 cities in the US over the past 15 years. The record of 20 was broken in week 15, when 24 deaths were reported. The numbers declined in weeks 16 and 17, but a new weekly record has been set for each of the past 5 weeks, even though lab confirmed cases have steadily declined to baseline levels as the flu season ended.

The record number of P&I deaths in El Paso has not been addressed by local or national agencies and these high levels are not reflected in lab confirmed cases raising concerns that the sampling and associated testing grossly under estimates the number of influenza deaths, particularly for H1N1. In the past 10 weeks the number of deaths in the 25-64 age group has been over represented, providing additional data linking the spike in deaths to unreported / undetected H1N1 infections, consistent with the linkage between the start of the increases and the outbreak of Chihuahua H1N1 across the border in Juarez, Mexico.

This outbreak lead to a pandemic alert, and the above data suggests that the cases are grossly under represented by the lab confirmed numbers. The pandemic alert was linked to the detection of the D225N receptor binding domain (RBD) change in the upper respiratory tract of severe and fatal cases, but sequences demonstrating a high frequency of D225N in Chihuahua have been withheld.

The withholding of these sequences, and the lack of comment on the record number of deaths in El Paso, continue to raise pandemic concerns.

Recently Swaziland has issued an H1N1 alert associated with 22 sympyomatic contacts of a fatal South African case. There have been two recent reports of H1N1 deaths in the Johannesburg Arrea (58M and 27M). One of these patients had arrived from Turkey. it is unclear if the H1N1 in Swaziland and South Africa is linked to the Chihuahua sub-clade in Mixico and South America, or is related to the sub-clade circulating in Singapore and Australia (which had also been circulating in Europe and the Middle East).

The record high number of P&I deaths in El Paso, as well as the increase in the US P&I rate to 7.20% in week 23, which is above the epidemic threshold, raises concerns that the dramatic spread of more virulent H1N1 will continue.

[dothedd]

South African H1N1 Deaths Raise Concerns
Recombinomics Commentary 18:30
June 16, 2011


They were on a flight from Istanbul to Joburg on May 18 when his condition worsened: “By the time we landed in Joburg, he was so ill that he couldn’t even stand up. His chest was so tight that he struggled to breathe,” his wife said.

She said that while her husband was being treated at the second hospital, a 27-year-old man had also died of swine flu-related complications.

The above comments describe two fatal H1N1 infections in a hospital in Pretoria/Johannesburg early in the flu season in the southern hemisphere. Since the first patient recovered prior to relapse and death it is unclead if the death was linked to the H1N1 in Turkey or a local H1N1 that was acquired at the hospital.

Similarly the relationship of the H1N1 in South Africa to the Chihuahua sub-clade in South America, or the Tamiflu/Relenza resistant H1N1 in Australia is unclear. Both sub-clades are rapidly spreading in the southern hemisphere and the Chihuahua sub-clade is associated with more severe outcomes inked to D225N. The sub-clade in Australia also has changes near the receptor binding domain (R208K, I219V), which may contribute to immunological escape and/or virulence.

One or both of the fatal cases appear to be linked to the 22 symptomatic contacts that precipitated the H1N1 alert issued by Swaziland.

Release of sequences and epidemiology associated with the fatal cases in South Africa would be useful.

[dothedd]

H1N1 Clonal Expansion of H274Y Tamiflu Resistance In Japan
Recombinomics Commentary 02:20
June 19, 2011

The recent examples of spread of Tamiflu resistance markers H274Y and S246N have raised concerns that resistance levels overall will increase, creating a genetic environment for the fixing of H274Y, as seen in seasonal H1N1 in the 2008/2009 season. These concerns were increased by the release of 2011 sequences from Japan.

These sequences also demonstrated the spread of H274Y by clonal expansion, and like the spread in Maryland and Delaware, the S188T sub-clade was involved. Although the geographical distinct expansions could also be distinguished by regional markers, both examples of expansion involved the dominant sub-clade in the northern hemisphere, confirming that the acquisition of H274Y did not impose a fitness penalty.

Similar results for S246N were seen in Singapore and Australia on a distinct but rapidly expanding sub-clade with R208K and I219V. The co-circulation of distinct sub-clades with H274Y or S246N raises concerns that recombination will put the same markers on the same gene as was seen in the fatal case in Australia, where the resistance synergized to reduce Tamiflu efficacy by 6000 fold.

The reported levels of H274Y are well below actual amounts. WHO consultants have agreed not to report mixtures with H274Y levels below 50%. However, such mixtures are widespread and quickly rise to levels above 50% as seen in the Perth case, A/Perth/29/2011, who was reported as H274Y positive 5 days after the start of Tamiflu treatment (initially the isolate, A/Perth/30/2010, had S246N, but was reported as wild type for position 274).

Although there were examples of H274Y and S246N clonal expansion 2009 and 2010, the number of transmissions was limited because of little immunity in the populations under 65, so there was little selection for individual sub-clades.

In 2011 the immunity levels are higher, leading to a small number of rapidly spreading sub-clones, like S188T, which is dominant. H274Y is now transmitting in multiple versions of the S188T sub-clade, including Delaware isolates which also have A189T or those in Japan with A200T. The acquisition of these additional receptor binding domain changes parallels the seasonal H1N1 evolution associated with the fixing of H274Y.

Thus, the fixing of H274Y in pandemic H1N1 is increasingly likely in the near term.

[dothedd]

WHO Inconsistent Comments on Japan H274Y Tamiflu Resistance
Recombinomics Commentary 16:40
June 19, 2011

Based on available data, among Japanese oseltamivir resistant viruses, the vast majority were from cases treated or prophylaxed with oseltamivir.

The above comments from the latest WHO report on oseltamivir (Tamiflu) resiistance in H1N1 implies that the increasing frequency of Tamiflu resistance (H274Y) reported in Japan is due to Tamiflu treatment, but the latest sequences released by Japan’s National Institute on Infectious Diseases (NIID) indicate H274Y increases are due to community clonal expansion across the entire country.

NIID has recently released 43 NA sequences collected between Dec 2, 2010 and Feb 2, 2011. 13 of these sequences (largely from Jan collections) had H274Y and this high frequency (30.2%) may be markedly above the reported 1.5% due to selection of samples for sequencing, but the published sequences clearly demonstrate clonal expansion because 10 of the 13 sequences are virtually identical to each other and form a phylogenetic branch which is exclusively populated by isolates with H274Y, Moreover, as seen by the list (below) of these 10 isolates, they are from eight different prefectures, indicating the community clonal expansion has spread throughout the country. This spread is supported by the spike in the detection frequency (to 4.8% in Feb and 5.8% in Mar), which would again be linked to clonal expansion and not linked to Tamiflu treatment (Table 1):

NOTE: Table 1 on the link below...

Thus, the basis for the WHO statement remains unclear. WHO consultants have agreed to report mixtures containing resistant sequences as wild type if the level of H274Y is below 50%, and therefore collection of samples after treatment may increase the level to more than 50%, leading to a resistance (H274Y) positive report. However, the virtual identity of the sequences from the 10 isolates below indicates the spread is via clonal expansion and not “spontaneous” mutations selected by Tamiflu treatment, as implied in the WHO statement above. Similarly, the wording of the WHO statement leaves open the possibility the patients were treated with Tamiflu after collection of the sample (which is common), and the classification of such patients as "Tamiflu treated" just adds to the confusion created by the “agreement” by consultants to not report Tamiflu resistance in samples where H274Y is below 50%.

In either case, the comments by WHO are grossly misleading and are little more than propaganda masquerading as science, creating the illusion that the increasing frequency of H274Y in community cases is linked to spontaneous mutations selected by Tamiflu use, rather than clonal expansion of a dominant sub-clade with H274Y.

A WHO explanation of these misleading reports is long overdue.

Closely related H274Y positive sequences:

A/HIROSHIMA/17/2011
A/WAKAYAMA/23/2011
A/SAPPORO/19/2011
A/NIIGATA/51/2011
A/HOKKAIDO/12/2011
A/YOKOHAMA/29/2011
A/NIIGATA/62/2011
A/KOBE/537/2011
A/YAMAGUCHI/35/2010
A/HIROSHIMA/49/2011

NOTE: TABLE 1

www.recombinomics.com/News/06191102/H1N1_H274Y_Japan_WHO.html

[dothedd]

Japan H274Y Tamiflu Resistance Clonal Expansion Concerns
Recombinomics Commentary 14:00
June 20, 2011

Based on available data, among Japanese oseltamivir resistant viruses, the vast majority were from cases treated or prophylaxed with oseltamivir.

The recently released H1N1 sequences from Japan clearly demonstrate the spread of oseltamivir resistance (H274Y) via clonal expansion. 10 of the 13 sequences with H274Y were closely related and mapped to a branch that had no isolates without H274Y. Moreover one additional sequence, A/Kagawa/2/2011, was closely related and was on an adjacent branch with one isolate, A/Shiga/35/2011, that did not have H274Y. One of the other isolates with H274Y, A/Iwate/1008/2011, was related to other sequences from Japan, while the other H274Y positive sequence, A/Hiroshima/45/2011, was related to US S188T sequences.

Thus, 12 of the 13 sequences were from 2011 (representing 38.7% of the 2011 sequences released by NIID) and the vast majority were spreading via clonal expansion, contracting the above quote in the most recent WHO update on H247Y in pandemic H1N1.

This pattern, which included a dominant sub-clade with H274Y, as well as additional examples of H274Y in the same sub-clade but on a distinct background, faithfully matches the pattern of H274Y in seasonal H1N1 in early 2008, which led to the fixing of H274Y in late 2008. In seasonal flu the pattern was independent of Tamiflu usage. H274Y was in patients who were not taking Tamiflu as well as countries where Tamiflu usage for treatment of seasonal H1n1 was minimal.

Thus, although the sequence data clearly demonstrates a pattern of clonal expansion of H274Y in pandemic H1N1 in Japan in 2011, the latest WHO report claims that such samples are from patients linked to Tamiflu usage. Since the expansion pattern covers the entire country and is clonal in nature, and the clone does not contain H274Y sensitive isolates, the WHO comments represent a glaring inconsistency.

The inconstancy is another example of WHO comments which are not supported by the sequence data generated by WHO affiliated labs, and like labs worldwide, describe the rapid appearance of H274Y in parients treated with Tamiflu, The short time frame between the start of treatment and the detection of H274Y signals spread via an H274Y mixture. The level of H274Y has now increased so the clonal expansion is clear, as is the lack of a requirement of Tamiflu treatement for the detection of this spread.

The remarkable parallels of the pattern in H274Y spread in seasonal H1N1 in 2008, and pandemic H1N1 in Japan in 2011 strongly predicts the fixing of H274Y in pandemic H1N1 in the near term.

Moreover, the WHO statement on the linkage to Tamiflu use represents a profound failure to communicate this clear clonal pattern that is not linked to Tamiflu usage, and raises significant competency concerns regarding the analysis of the evolution of pandemic H1N1 by WHO consultants and communicators.

[dothedd]

WHO Comment On H274Y Tamiflu Resistance Raises Concerns
Recombinomics Commentary 12:00
June 21, 2011

Based on available data, among Japanese oseltamivir resistant viruses, the vast majority were from cases treated or prophylaxed with oseltamivir.

The recently release sequences by the US CDC and Japan’s NIID provided clear examples of H274Y (oseltamivir / Tamiflu resistance) clonal expansion in the United States and Japan. The US isolates were geographically clustered in Delaware and Maryland, with four isolates each. These isolates formed small clusters of clonally expanding H274Y. Seven of the eight were in the S188T sub-clade. The first three isolates from Delaware had A189T appended onto this background. A189T is the receptor binding domain change in the Chihuahua sub-clade and the only public Chihuahua sequence with H274Y is also in Maryland. Thus, the US sequences demonstrated the movement of H274Y onto geographically clustered genetic backgrounds due to recombination driven by a high concentration of H274Y.

The sequences from Japan represented the converse situation, where the same sub-clade, which was also S188T, had spread throughout the country. This sub-clade had A200T appended onto the S188T background and 10 fo the 13 sequences for a branch which was exclusively populated by sequences with H274Y. This expansion and distribution closely mirrored the spread of H274Y in seasonal H1N1, where H274Y was in clade 2B (represented by Brisbane/59/2007) and most isolates were closely related and matched the initial sequences from northern Europe, but H274Y was also present in smaller clusters which were clade 2B, but slightly different than the larger cluster, reflecting additional recombination leading the spread of H274Y in closely related sub-clades.

The striking parallels between H274Y spread in seasonal H1N1 in 2008 and pandemic H1N1 in 2011 strongly suggests that H274Y will soon become fixed in pandemic H1N1. Like 2008, the spread is driven by clonal expansion and not selection through Tamiflu treatment, which is why the latest comment by WHO on sequences in Japan is cause for concern. The clear clonal expansion is ignored in the WHO update, and instead reference is made to a linkage between the detection of H274Y and use of Tamiflu by the corresponding patients. This disconnect between the sequence data and the WHO announcement raises serious competency concerns regarding those responsible for this conclusion / announcement.

The lessons learned from the fixing of H274Y in seasonal H1N1 were clear and the failure to relate this pattern to pandemic H1N1 is alarming.

An explanation by WHO of the data and rationale behind their bizarre announcement (stated above) is needed, along with an independent scientific investigation of the underlying data and personnel responsible for this gross misinterpretation or misleading comment linked to very clear cut sequence data.

www.recombinomics.com/News/06211101/H1N1_H274Y_WHO_Concerns.html

[dothedd]

Withheld CDC H274Y H1N1 Tamiflu Resistance Sequences (06/30/11 20:55)

S246N and H274Y Tamiflu Resistance Combination Concerns (06/30/11/14:30)

D225N On S186P Background In Chihuahua Mexico (06/24/11 00:40)

H1N1 RBD Changes - D225N and Q226R In Egg Isolates (06/23/11 22:40)

www.recombinomics.com/whats_new.html

[dothedd]

Alarming Increase In H1N1 Low Reactors In Japan
Recombinomics Commentary 23:55
July 1, 2011


Today NIID released 37 HA sequences from 2011 (at GISAID), which were associated with frequent Tamifu resistance (H274Y) in most associated NA sequences, as well as one NA sequence with S246N, signaling additional clonal expansion.

However, the HA sequences had an alarming number of changes at positions 156-159, with 6,1,9,2 changes respectively. The prior data on 33 HA sequences from Japan had postion 156-159 chnages of 1,3,5,1, respectively. Changes at these positions are linked to low reactors which have titers reduced four fold or more when tested against the current H1N1 vaccine target, A/California/07/2009. These numbers are likely to represent an underestimate because the isolates involved growth on mammalian MDCK cells, which are rich in gal 2,6 gal. Isolation with eggs, which are rich in 2,3 gal receptors would have selected addition examples of changes at this position. These changes are biologically and clinically relevant because they are associated with vaccine escape as well as preferential growth in human lung, which is rich in gal 2,3 receptors.

The changes are increasing in prevalence, but these alarming changes are being discounted with claims that the changes are artifacts of culture since detection is more difficult in direct sequencing of clinical samples since the changes are frequently present as mixtures with wild type.

The discounting of these changes has significant consequences. In the United States Pneumonia and Influenza (P&I) deaths were at record levels in 2011 and were similar to rates recorded in 2008. However, in 2008 the vaccine mismatches with all three vaccine target were eventually acknowledged and all three targets were updated for the following season. In contrast, in 2011 claims were made that all three targets were “matches” due in part to extensive manipulation of laboratory test results, including claims that negative data invalidated positive data. Consequently, all three vaccine targets remained unchanged for the 2011/2012 season.

In addition to the elevated P&I death rate, the frequency of Tamiflu resistance has also increased and significant clonal expansion has been seen in the United States and Japan for H274Y. The reported numbers are underestimates because WHO collaborating centers have agreed to not report Tamiflu resistance when H274Y levels are less than 50% of the sample signal. In addition, clonal expansion of Tamiflu and Relenza resistance (S246N) has been reported in Singapore and Australia, including 30% of H1N1 isolates from Darwin.

These increases in low reactors as well as Tamiflu resistance highlight the folly of lab manipulations to generate under-estimates for “scientific” public reports published by government and international agencies, and these policies and publications remain hazardous to the world’s health.

[dothedd]

More H1N1 Low Reactors In Japan Raises Vaccine Concerns (08/08/11 18:30)

trH3N2 Detection and Reporting Delays Rasie Concerns (08/06/11 20:30)

Minnesota trH3N2 and Huron Ohio Fair trH1N1 Clusters Linked (08/06/11 17:00)

Pandemic trH3N2 Vaccine Linked To Minnesota Cluster (08/06/11 14:30)

New trH3N2 Case Raises Pandemic Concerns (08/04/11 18:05)

Novel Influenza Case In MMWR Week 30 (08/04/11 14:05)

Vaccine Pandemic Concerns (08/04/11 04:05)

CDC Testing Three New Influenza B Vaccine Targets (08/03/11 23:55)

CDC Testing Two New Seasonal H3N2 Vaccine Targets (08/-3/11 23:20)

CDC Testing Pandemic trH3N2 Vaccine (08/03/11 21:50)

CDC Testing New H1N1 Vaccine Target (08/03/11 18:30)

Identical H5N1 Deletion In Indonesia Egypt and China (07/25/11 15:00)

Influenza Vaccine Concerns (07/21/11 15:30)

H1N1 S188T Dominant In Southern Hemisphere (07/20/11 18:10)

LINK for the above articles below:

www.recombinomics.com/whats_new.html

[dothedd]

D225G In Fatal Chihuahua H1N1 Index Case
Recombinomics Commentary 18:30
August 17, 2011


The CDC has released another series of 2011 H1N1 sequences. Included was A/Mexico/2058/2011 from a 36M collected on March 15, 2011. The age, gender, and collection date match an earlier case which was associated with another sequence, A/Mexico/DRE1945/2011. However, it is unclear why sequences from the same patient would be given different numbers. The March 15 was a week earlier than subsequent collections, strongly suggesting that Mexico/2058 and Mexico/DRE1945 are from the first confirmed cases in Chihuahua from the Chihuahua sub-clade.

The sequences from the National Labs contained D225N, which was stated in the title of the associated characterization sheet, “Detection of D222N substitution in Pandemic Influenza A (H1N1) Virus in Chihuahua, Mexico”. The CDC sequence was from an egg isolate, and was closely related to the earlier sequence, but had D225G, raising concerns that the frequency of D225G is grossly under-reported because most isolates are from MDCK cells which select against D225G.

The sequence from the lab in Mexico was direct and from a sample from the upper respiratory tract and was not reported as a mixture. Similarly, the CDC sequence was from an egg isolate, and also not reported as a mixture, although the age, gender, and collection date indicate both sequences were from the first confirmed case, which was fatal and from the partner of another fatal case, who had similar symptoms, but isolation attempts failed. The linkage of these two fatal cases, as well as additional severe and fatal cases from the same traffic patrol office led to a WHO pandemic alert.

The time delay, and egg isolation raises concerns that there are many more sequences from Chihuahua with D225G and D225N, which would provide more linkage to severe and fatal H1N1 cases, as indicated in multiple anecdotal reports. Similarly, the detection of D225G in an egg isolate raises concerns that the H1N1 sequence database in general grossly underestimates the frequency of D225G and D225N in severe and fatal cases since most sequences are from isolates grown in mammalian MDCK cells.

The changes in positions 225 and 226, as well as positions 156-159 are important because they are associated with immunological escape, as well as targeting of human lung. Both human lung and chicken embryos in eggs, are rich in gal 2,3 receptors.

The heavy use of MDCK would also produce a gross undercount in low reactors, which led to the recent recommendation to leave all three vaccine targets unchanged in spite of record high fatality rates in pneumonia and influenza (P&I) patients. These concerns are likely linked to the recent sequences for new vaccine targets. The latest, A/Victoria/502/2010, was just released by the WHO regional center in Australia. Like the new pandemic H1N1 target released by the CDC, it has Q226R. The initial egg isolate from this patient had D225G, while an MDCK isolate was wild type at positions 225 and 226. These new vaccine target raise concerns that the current vaccine in use in the southern hemisphere, as well as the vaccine shipping for the 2011/2012 season in the northern hemisphere, will have limited utility.

[dothedd]

Chihuahua H1N1 LOW REACTOR In Alaska
Recombinomics Commentary 23:55
August 17, 2011


The CDC recently released a Chihuahua sub-clade sequence, A/Alaska/14/2011, which they designated a LOW REACTOR (at GISAID). The HA sequence (from 41M collected March 30, 2011) had a mixture at position 159 (N159D and N159E) and both changes have been noted in other low reactors. In the 2009/2010 season all five US H1N1 sequences designated as low reactors by the CDC had a change at position 159.

The sequence was closely related to another Chihuahua sequences from Alaska, A/Alaska/12/2011, which was collected 6 days later from a child (12M), raising the possibility that cases were linked. Both sequences were deposited June 21, but the release of the low reactor sequence was delayed almost two months (released August 15) for unknown reasons. However, the sequence similarities and collection dates raise concerns that N159D and N159E are also present in the latter collection, but not detected.

The failure to identify changes at positions 156-159 has becme an issue after the CDC complained about other WHO labs (Mill Hill in the UK, NIID in Japan, WHO regional center in Australia) were finding too many changes at these positions because they didn’t use “normal” MDCK cells. Changes at these positions create LOW REACTORS like the CDC designated sequence above, but the CDC identified a very low number of such cases (as noted, in 2009/2010 no US isolates without a change at position 159 were designated as low reactors).

The absence of low reactors led to the unanimous decision to leave all three vaccine targets unchanged for the 2011/2012 season. However, the CDC and WHO region center have recently begun testing new candidates (three for influenza B, three for seasonal H3N2, and two for pandemic H1N1. For pandemic H1N1, both candidates have Q226R, although A/South Carolina/02/2010 and A/Victoria/502/2010 have representations without Q226R (egg isolates of Victoria/502/2010 have D225G). In addition to the receptor binding domain changes, the US vaccine target, A/South Carolina/02/2010, also has K157E, even though the original sequence was wild type. The inclusion of K157E and Q226R in the new H1N1 vaccine targets raises concerns that these levels of these LOW REACTOR changes or receptor binding domain changes are markedly higher than the levels indicated in the sequences from H1N1 grown on “normal” MDCK cells.

[dothedd]

WHO Testing New Pandemic H1N1 Vaccine Target
Recombinomics Commentary 13:30
August 18, 2011


Recently released H1N1 sequences (at GISAID) by the WHO regional center in Australia included a new pandemic H1N1 target, IVR-159 A/Victoria/502/2010. The HA sequence included Q226R, which was also added to the H1N1 vaccine target being tested by the CDC, A/South Carolina/02/2010. Q226R was not in either original sequence. This receptor binding domain change increases growth in chicken eggs, and may be grossly under-represented in the GISAID and Genbank sequence databases because most H1N1 isolates are from infections of mammalian cells (MDCK).

The use of MDCK cells reduces the frequency of changes at key HA positions, such as 225 and 226 in the receptor binding domain, as well as positions 156-159 which are involved in immune recognition. The new CDC vaccine target also had K157E and two way testing using the old and new vaccine targets produced “LOW FAIL” results indicating the anti-sera against the old target, A/California/07/2009, had limited activity against the new target, and anti-sera against the new target had limited activity against the old target.

Last year an egg isolate of A/Victoria/502/2010 had D225G. The new vaccine target is wild type at position 225, but has Q226R.

The testing of new targets at this time is unusual because at the most recent meetings of the vaccine advisory committee for the US FDA, as well as the WHO meeting, recommended no changes in the three vaccine targets, and that vaccine for the 2011/2012 flu season in the northern hemisphere will be shipping shortly.


The testing of new targets by the CDC and WHO at this time raises serious concerns that the vaccine for the 2011/2012 season will have limited utility.

[dothedd]

WHO Testing New Seasonal H3N2 Vaccine Target
Recombinomics Commentary 15:55
August 18, 2011


Recently released H3N2 sequences by the WHO regional center in Australia included a new seasonal H3N2 target, A/Victoria/563/2010 IVR-160. This sequence follows the release of three H3N2 targets by the CDC, A/Brisbane/11/2010 X197, A/Rhode Island/01/2010 X-199, and A/Perth/10/2010 X-207. These four targets are being tested as a replacement for the current H3N2 target, A/Perth/16/2009.

The testing of new targets at this time is unusual because at the most recent meetings of the vaccine advisory committees for the US FDA, as well as the WHO meeting, the committees recommended no changes in the three vaccine targets, and that vaccine for the 2011/2012 flu season in the northern hemisphere will be shipping shortly.

The testing of new targets (seasonal H3N2, pandemic H1N1, and influenza B) by the CDC and WHO at this time raises serious concerns that the vaccine for the 2011/2012 season will have limited utility.

[dothedd]

H274Y Tamiflu Resistance Clonal Expansion - Newcastle Australia Recombinomics Commentary 21:40
August 26, 2011


A cluster of oseltamivir-resistant A(H1N1)2009 influenza cases with onset between May and August 2011 has been detected in the Hunter region of New South Wales (NSW), Australia.

Viruses from 25 of 184 (14 percent) A(H1N1)2009 cases from the Hunter New England region exhibited highly reduced oseltamivir sensitivity due to the H275Y substitution in the neuraminidase.

15 of the 1st 16 cases lived within a 50-km radius of the regional
centre of Newcastle. 16 of the 25 patients have been interviewed, and none had received oseltamivir prior to influenza specimen collection.

The above comments describe the clonal expansion of H274Y in New South Wales province in Australia. The WHO regional center in Australia recently released three sequences (A/NEWCASTLE/14/2011, A/NEWCASTLE/47/2011, A/NEWCASTLE/58/20110 from Eleebana, Rutherford, and Cessnock, which are all within 30 km of Newcastle, and collected in June and July by John Hunter Hospital, Virology Unit, Clinical Microbiology in Lambton.

The HA from these sequences had S188T and all three sequence were identical, but none had H274Y. However, the identity of all three sequence from the region with H274Y suggests the resistant H1N1 matches the above sequences with H274Y appended to that genetic background.

These sequences are distinct of the clonal expansion of H274Y in Japan, or Delaware and Maryland, which involve additional S188T sub-clades. The expansion of multiple sub-clade with S188T and H274Y in three different continents indicates H274Y on an S188T is evolutionary fit and is rapidly spreading via hitchhiking and clonal expansion.

[dothedd]

Indiana trH3N2 Case With Pandemic H1N1 MP
Recombinomics Commentary 14:20
August 27, 2011


The CDC has released a full set of pandemic H3N2 (trH3N2) sequences (at GISAID) from a toddler (1M) collected July 24, 2011 in Indiana (A/Indiana/08/2011). The isolate is a reassortant with a pandemic H1N1 MP, which is the first such constellation reported in humans. Moreover, the PB1 is most closely related to the PB1 from the 2007 Huron County Fair and therefore does not have E618D.

The H3 sequence is closely related to the 2010 human cases (A/Minnesota/11/2010, A/Wisconsin/12/2010, A/Pennsylvania/40/2010, A/Minnesota/09/2010), but is most closely related to two recent swine isolates, including one from Indiana (A/swine/North Carolina/A01049436/2011 and A/swine/Indiana/A0109091/2010). However, the MP in the two swine isolates are identical, but related to North America swine, which is easily distinguished from the pandemic H1N1 MP in the above case.

The presence of pandemic MP in the recent cases raises concerns a further adapotation of trH3N2 to humans.

The CDC has begun testing a trH3N2 vaccine target, A/Minnesota/11/2010 X-203, which is related to the above case and from a cluster.

The latest case is not described in the CDC's MMWR or website. More detail on those case and symtoms in contacts would be useful.

[dothedd]

trH3N2 Sequence Cluster Raises Pandemic Concerns
Recombinomics Commentary 14:20
August 29, 2011


The recently released Indiana trH3N2 HA sequence, A/Indiana/08/2011, is closely related to multiple 2010 human trH3N2isolates (A/Wisconsin/12/2010, A/Pennsylvania/40/2010, A/Minnesota/11/2010), and the collection date of July 24, 2011 suggests it may the trH3N2 case cited in the week 30 MMWR on August 5, 2011. The CDC has not updated its website on recent human cases involving trH3N2, and has not provided any detail on this case (1M), other than collection date, age, and gender on the sequence characterization sheet..

However, the close relationship to the earlier isolates above raises concerns that this H3 sequence is transmitting in humans. The above Wisconsin and Pennsylvania cases were symptomatic within a week of each other (September, 2010), raising concerns that a sub-clade with this H3 sequence was transmitting among humans last fall. This concern was increased by the H3 sequence of the Minnesota isolate (November, 2011), which was virtually identical to the two earlier isolates. The concern was increased further by the report indicating the daughter of the Minnesota case (31M) was also serlogically confirmed to have been trH3N2 infected (without swine contact), and additional contacts had ILI (influenza like illness).

The recent selection of the sequences of the index case as a potential trH3N2 vaccine target added to these concerns. All of the earlier cases were due to infections in late 2010. The Indiana sequence demonstrates the continued circulation of this H3 sequence in humans. The H3 sequence has a cluster of six consecutive polymorphisms (G855A, C866A, G872A, C873T, A874A, and C876A) which are present in all four human sequences, as well as two recent swine sequences (A/swine/North Carolina/A01049436/2011 and A/swine/Indiana/A0109091/2010) generated by the Veterinary Diagnostic and Production Animal Medicine at Iowa State University. This department and the USDA at ISU have markedly increased the number of recent swine H3 sequences, and the latest sequences demonstrate the preferential clustering of this H3 related sequence in human isolates.

This clustering increases concerns regarding human transmission. The latest human isolate from Indiana is a reassortant. Although the H3 and N2 sequences are closely related to the swine sequences, the MP sequence matches the pandemic H1N1 sub-clade, signaling an independent introduction of this isolate into the human population. The Indiana isolate is the first example of a human trH3N2 with a pandemic H1N1 gene.

However, although this isolate represents a new introduction, the close H3 match with the three earlier sequences (and trH3N2 linked to the MN cluster), indicates this H3 is linked to more efficient transmission of trH3N2 in humans.

More information on the Indiana case and symptomatic contacts would be useful.

[dothedd]

BELOW IS THE LINK TO THE INDIVIDUAL "News Commentary"

www.recombinomics.com/whats_new.html


Linkage of trH3N2 Cases To Washington County Fair Is Tenuous (09/08/11 04:40)

Severe and Widespread trH3N2 Raises Concerns (09/07/11 21:15)

CDC Release Washington County PA trH3N2 Sequences (09/07/11 19"45)

Pennsylvania and Indiana trH3N2 Matches Signal Pandemic (09/07/11 18:00)

CDC trH3N2 Testing Bias Raises Pandemic Concerns (09/07/11 02:15)

CDC Comments On trH3N2 Cluster Raise Concerns (09/06/11 21:20)

Media Myths On trH3N2 Washington County Fair Cluster (09/06/11 18:00)

Washington Fair trH3N2 Cluster Signals Human Transmission (09/05/11 21:00)

Is the trH3N2 Case In Pennsylvania Unsubtypable? (09/05/11 19:40)

Evolution of the 2011 Swine trH3N2 Pandemic (09/05/11 13:50)

CDC Unsubtypable For Week 33 Raises trH3N2 Pandemic Concerns (09/05/11 05:15)

CDC Requests Nasopharyngeal Swabs for trH3N2 Testing (09/04/11 21:30)

Frequent H1N1 M Gene Acquistions In North American Swine (09/04/11 14:00)

H1N1 M Gene Critical In Human Transmission - trH3N2 Concerns (09/03/11 19:30)

CDC Comments On trH3N2 Match in PA and IN cases (09/03/11 17:50)

Pennsylvania trH3N2 Testing Symptomatic Fair Attendees (09/03/11 12:50)

CDC Hiding trH3N2 Cluster In Minnesota (09/03/11 10:50)

trH3N2 Match in PA and IN Raises Pandemic Concerns (09/02/11/04)

trH3N2 Cases In Indiana and Pennsylvania Match (09/02/11 18:50)

CDC Silence On Spike In trH3N2 Cases Raises Concerns (09/02/11 14:05)

Two More trH3N2 Cases (IN & PA) Raise Pandemic Concerns (09/02/11 02:10)

FAO Warns of Fujian H5N1 Spread in China and Vietnam (08/29/11 15:00)

[dothedd]

Lack of Contact Between trH3N2 Cases and Swine
Recombinomics Commentary 22:15
September 8, 2011


No direct exposure to swine was identified for this child; however, a caretaker reported direct contact with asymptomatic swine in the weeks before the boy's illness onset and provided care to the child 2 days before illness onset. No respiratory illness was identified in any of the child's family or close contacts, the boy's caretaker, or in the family or contacts of the caretaker.

All three of the patients were in the area where swine were exhibited and one had direct contact with swine.

The above comments described the four recent confirmed trH3N2 cases in Indiana and Pennsylvania. As noted above, contact with swine was only identified for one of the four cases (2F from Schuylkill County who visited the fair on August 16). Sequence data released yesterday by the CDC indicated that the trH3N2 isolates from each patient were virtually identical, even though there has been no reported epidemiological link between the cases. The boy, A/Indiana/08/2011, was infected in Indiana in July, while the collection dates for samples from the two girls were August 25, A/Pennsylvania/11/2011, and August 26 (previously reported as July 27), A/Pennsylvania/10/2011, well after the last day of the fair, August 20. The samples were initially tested by the University of Pittsburgh Medical Center or the Pennsylvania Department of Health, respectively, suggesting independent infections. The sequences from the Schuylkill patient had the same constellation of genes that had drifted slightly from the other three, again supporting an independent introduction and source.

However, although all four patients appear to have been infected independently, they were all infected by a trH3N2 related to earlier triple reassortant infections in humans. Five of the genes are closely related to the 2010 trH3N2 infections in Wisconsin (A/Wisconsin/12/2010), Pennsylvania (A/Pennsylvania/40/2010) and a cluster in Minnesota (A/Minnesota/11/2010). However, the new constellation has acquired a PB1 that is closely related to sequences from the tr H1N1 Huron Fair cluster (A/Ohio/01/2007 and A/Ohio/02/2007), an NA closely related to another trH3N2 Pennsylvania case, A/Pennsylvania/14/2010, and an M gene segment closely related to pandemic H1N1.

Thus, the latest trH3N2 is the most common trH3N2 found in humans in 2010, which has acquired three gene segments previously linked to trH3N2 or trH1N1 human cases, signaling human adaptation, which includes the M gene segment which is critical for transmission in humans.

The identifies between the trH3N2 isolates from independent infections in Indiana and Pennsylvania, coupled with the acquisition of the M gene segment from pandemic H1N1, raises concerns that the trH3N2 is efficiently transmitting, especially in children, and is largely undetected because trH3N2 is largely limited to patients with a perceived or possible swine connection, which is reinforced the CDC request for samples from patients with flu-like symptoms who are exposed to swine.

Thus narrow search, will delay detection of widespread trrH3N2 which is expected based on the acquisition of H1N1 M gene and the identities between the four confirmed cases in Indiana and Pennsylvania.

[dothedd]

Commentary

Silent Spread of trH3N2 Raises Concerns
Recombinomics Commentary 13:00
September 9, 2011


No direct exposure to swine was identified for this child; however, a caretaker reported direct contact with asymptomatic swine in the weeks before the boy's illness onset and provided care to the child 2 days before illness onset. No respiratory illness was identified in any of the child's family or close contacts, the boy's caretaker, or in the family or contacts of the caretaker.

The above comments from today’s MMWR have been cited as another example of human to human spread because the confirmed case had no contact with swine. The interaction of the caretaker with swine is cited as a potential source of the infection, which would signal silent spread since the caretaker was asymptomatic, as were the swine. Thus, the link between the confirmed cases and swine is purely speculative, because no symptoms were reported in contacts, and no trH3N2 was isolated from the swine or any contacts.

However, the sequences of the eight gene segments from the case (2M), A/Indiana/08/2011, defines a constellation of flu genes that has never been reported in swine. Moreover, virtually identical sequences were found in two cases (both 9F) in Washington County, Pennsylvania (A/Pennsylvania/10/2011 and A/Pennsylvania/11/2011), as well as a closely related drift variant, A/Pennsylvania/09/2011, from a case (2F) from Schuylkill County in eastern Pennsylvania, who visited the western Pennsylvania fair on August 16. However, like the case in Indiana, no symptomatic swine have been identified and no contacts have been lab confirmed.

All four of these isolates have the same constellation of genes, including those from 2010 human cases in Pennsylvania, Wisconsin, and Minnesota. The Minnesota isolate. A/Minnesota/11/2010, was from the father of a second case who was trH3N2 lab confirmed, but had no contact with swine, representing another example of human to human transmission (and additional family members had flu-like symptoms, but serological testing was inconclusive).

These examples of human to human transmission, absence of swine contact in most of the recent cases, as well as the recent acquisition of the pandemic H1N1 M gene, which is critical for efficient human to human transmission, strongly suggest that the recent cases were not due to transmission from swine. The two most recent cases had no known contact with swine and samples were collected five or six days after the Washington county fair ended, casting further doubt on a swine source, The linkage to swine is largely linked to testing, since trH3N2 testing is focused on patients with swine association, as indicated in the latest CDC request for samples.

This requirement for a swine link has no scientific basis, and heavily biases the data, since symptomatic patients are rarely tested for trH3N2 if they have no swine connection. Instead these patients test positive for seasonal H3N2 because only an H3 sub-typing test is run, which has previously been positive for trH3N2 cases since the H3 and N2 trace back to a human infection of swine in the 1990’s (or 2003 for some N2 sequences). However, the recently reported unsubtypable in week 33 in the updated FluView table suggests that in some instances the trH3N2 infections will not subtype. The case from Schuylkill County represents the only confirmed trH3N2 collection in week 33, suggesting the H3 has evolved far enough away from the determinants recognized by the H3 typing reagents to produce an negative result in an influenza A positive sample. The 2F case was influenza A positive, but confirmed with a PCR test directed against swine sequences.

Thus, prior and recent H3N2 cases should be PCR tested to begin to gain an understanding of the extent of trH3N2 spread.

Currently Pennsylvania has the largest number of trH3n2 confirmed cases (five) and reported a large number of unsubtypables in the 2010/2011 season. A retesting of these samples, as well as recent influenza A positive samples, with emphasis on those from children, would be useful.

[dothedd]

Unsubtypables and trH3N2 Surveillance
Recombinomics Commentary 19:00
September 9, 2011


One case of human infection with a novel influenza A virus was reported by the Pennsylvania Department of Health. The patient was infected with a swine origin influenza A (H3N2) virus. The patient reported contact with pigs in the week preceding symptom onset on September 6, 2010, did not require hospitalization, and has since fully recovered. Initial testing of the specimen indicated a seasonal influenza A (H3N2) virus and the specimen was submitted to CDC as a routine surveillance sample. The delay from onset to detection occurred because attempts to culture the virus were unsuccessful. RT-PCR testing confirmed swine-origin influenza A (H3N2).

The above comments from the week 4 FluView describe an earlier isolate from Pennsylvania, A/Pennsylvania/40/2010 (PA/40/10), which is closely related to A/Wisconsin/12/2010 and A/Minnesota/11/2010, which are precursors to the four confirmed trH3N2 2011 cases. As noted above, the PA/40/10 sample was initially classified as seasonal H3N2 and the announcement of trH3N2 was made 5 months after collection.

The H3 sequences from the latest trH3N2 isolates has evolved further from the 2010 sequences, and the unsubtypables listed for week 33 and week34 the week 35 report are likely A/Pennsylvania/09/2011 (PA/09/11) and A/Pennsylvania/11/2011 (PA/11/11) and suggests that the more 2011 trH3N2 isolates will register as influenza A positive, but H3 and H1 negative, which would facilitate detection by state labs who routinely determine sub-types.

However, as seen in the week 35 report, many samples are not sub-typed, and the other two recent isolates may be in that category, but made be subsequently added after sub-type failure (and associated confirmation of trH3N2).

Thus, the comments posted above, coupled with the concentration of confirmed trH3n2 cases in Pennsylvania and prior reports of unsubtypable samples warrant a review / retesting of those samples via swine specific PCR analysis, as was done with PA/09/11.

This review should identify many additional prior trH3N2 cases in the 2010/2011 season and focus testing on influenza A positive cases, including those without linkage to swine.

[dothedd]

Unsubtypables Raise trH3N2 Pandemic Concerns
Recombinomics Commentary 02:20
September 10, 2011


The graph below has the CDC’s week 35 FluView, which shows a low level of influenza activity in recent weeks, which is expected since the flu season doesn’t begin until week 40 in the United States. The expanded view of recent weeks includes the unsubtypable isolates for week 33 and week 34. The week 33 unsubtypable was expected, because last weekend the designation of the case was added to the underlying data. It was assumed that the data reflected A/Pennsylvania/09/2011, which was isolated from a sample collected August 20, the last day of week 33. The appearance of a second unsubtypable confirms that these cases are from the Washington County cluster, and the week 34 unsubtypable represents either A/Pennsylvania/11/2011 collected August 25, or A/Pennsylvania/10/2011, collected August 26, since all three H3 sequences are virtually identical and match A/Indiana/08/2011.

The presence of only two of the four cases is likely due to the number that has been conclusively shown to be negative in the H3 sub-typing assay or the typing of some as seasonal H3N2. Thus, the number of unsubtypable may increase in upcoming weeks as the samples are more fully tested.

NOTE THE GRAPH ON THE LINK BELOW....

The graph also includes the latest data, week 35, which had 10 positive samples, all of which were influenza A. However, only two were sub-typed (pandemic H1N1 and seasonal H3N2). The low frequency of samples sub-typed may reflect additional cases that are unsubtypable. These would represent new trH3N2 cases because the earlier trH3N2 samples were collected in weeks 30 and 34. The absence of an unsubtypable in week 30 may indicate A/Indiana/08/2011 has not been added yet or is not included in samples that are reported by FluView. Thus, many of the p"not suntyped" by in fact be trH3N2 while others designated as seasonal H3N2 may in fact be trH3N2, as was seen for A/Pennsylvania/40/2010.

Thus, while all recent unsubtypables may be trH3N2, all trH3N2’s may not register as unsubtypables. Therefore, these recent isolates that were designated as seasonal H3N2 should be PCR tested to see if they are really trH3N2.

Concerns that recent influenza A positives are trH3N2 are increased by the lack of sub-typing for the week 35 isolates, where only 2 of the 10 samples have been sub-typed. Thus, all influenza A samples should be PCR tested to determine the true frequency of trH3N2 cases circulating in the United States.

This summer the CDC has released five recent H3N2 sequences from the US, and four of the five are trH3N2.

LINK:
www.recombinomics.com/News/09101101/trH3N2_Unsub_Pandemic.html

[dothedd]

H3N2 Dominance In US Raises trH3N2 Pandemic Concerns
Recombinomics Commentary 12:45
September 10, 2011


The figure below represents the CDC’s week 35 FluView graph of serotypes. In addition to the unsubtypable for week 33, which was added to the underlying data a week ago, the updated data includes an unsubtypable for week 34 as well as two additional H3N2 isolates. These updates after the initial report are not unusual, but the increases in H3N2 raise concerns that many or most of these isolates being reported as seasonal H3N2 are actually trH3N2.

NOTE CHART IN BELOW LINK


In the past three weeks H3N2 has become the dominant serotype, as represented by the seasonal H3N2 column as well as the unsubtypables, which are clearly trH3N2 isolates which correlate with the reported case from Washington County, Pennsylvania.
As was reported for the delayed reporting of an early trH3N2 isolate from Pennsylvania, A/Pennsylvania/40/2010, trH3N2 isolates frequently subtype as seasonal H3N2 because the H3 and H2 trace back to reasortants involving seasonal H3N2. Thus, the human origin of these genes generates a positive result when assayed by H3 and N2 typing reagents, which target human determinants.

However, the two recent unsubtypables indicate that the 2011 isolates from Pennsylvania fail to type, leading to a sub-typing profile of influenza A positive but H3 and H1 negative for trH3N2, similar to results for pandemic H1N1, which has a swine H1 and N1. Thus, the recent evolution of H3 has moved the trH3 away from recognition by human H3 reagents, but other isolates, like A/Pennsylvania/40/2010 will return a seasonal H3N2 result. Thus, many of the recently reported seasonal H3N2 isolates may be trH3N2.

These newly reported H3N2 isolates, as well as those that are influenza A positive, should be PCR tested, which is also true for early H3N2 isolates, including the large number of unsubtypables reported by Pennsylvania in the 2010/2011 season.

More detail on the recent H3N2 isolates classified as seasonal H3N2 would be useful.

LINK TO CHART:

www.recombinomics.com/News/09101102/trH3N2_H3N2_Pandemic.html

[dothedd]

Unsubtypables Dictate Widespread trH3N2 PCR Testing
Recombinomics Commentary 13:45
September 11, 2011


The latest CDC FluView (week 35) with two unsubtypables (unable to subtype) highlights the dramatic recent changes in the H3 sequences in the 2011 trH3N2 isolates, which include R50G, S107T, T117N, D133G, E142G, H156Y, A163E, L164Q, R189K, T203I, P274H, G276D, N277E, R299K, L427I, N444S, K452R, V489I, D492N.

These changes create an “unsubtypable” result because the changes are too great for recognition by the reagents used to subtype human H3. However, most of the above changes are also in the closely related 2010 trH3N2 isolates, like A/Pennsylvania/40/2010, which did initially type as human seasonal H3. This variability raises concerns that some trH3N2 will sub-type as seasonal H3N2, while others will be usubtypable as seen with two of the four 2010 isolates which have H3 sequences that are virtually identical.

Most of those trH3N2 cases that type as seasonal H3N2 will not be further classified. A small sub-set will be characterized furth in antigen characterization tests (which would identify such cases as LOW REACTORS) or sequencing, which would easily identify the above changes as well as a similar number of synonymous change. Thus, even partial sequences will easily distinguish seasonal H3N2 from swine trH3N2. However, these test generally rely on the isolation of virus and are done by the CDC, so the number of such identification will be low and may be significantly delayed, as seen in the 5 month delay for A/Pennsylvania/40/2010. However, if the pandemic trH3N2 outcompetes the seasonal H3N2, these more complicated tests will clearly identify trH3N2, but the lag time may be significant.

PCR testing for the swine evolved sequences is very rapid and quickly identifies trH3N2, as was seen for the four 2011 sequences, and the PCR testing can be done by the state labs. Therefore, such screening should be dramatically increased, as was done for pandemic H1N1.
The PCR testing would quickly identify the extent of the trH3N2 spread, which is likely to present in patients without swine exposure, as was seen in 3 of the 4 recent cases, as well as patients who do not live in areas with swine, who are rarely tested for trH3N2.


The identities between the isolates in Indiana and Pennsylvania strongly suggest this latest version of trH3N2 is spreading in humans since it has acquired the pandemic H1N1 M gene segment, and the H3 and other internal genes cluster in human trH3N2 cases, which is easily seen in phylogenetic analysis.

Release of results of widespread PCR testing on influenza A positive samples would be useful.

[dothedd]

The New Contagion trH3N2
Recombinomics Commentary 15:30
September 11, 2011


The latest CDC FluView (week35) coincides with the release of the movie “Contagion” and it is worth comparing trH3N2 to the virus in the film, because both are new contagions. In the movie the agent (MEV) was a recombinant with sequences from bat and swine, and as seen in the samples frozen in liquid nitrogen, the MEV isolates are in the same rack as SARS and H1N1, which is appropriate since SARS is thought to reside in bats, while H1N1 is a triple reassortant of swine origin.

The spread of MEV parallels the SARS CoV, which launched its international spread in Hong Kong. A physician who had been treating SARS patients in Guangdong Province came to Hong Kong for a wedding. He stayed at the Metropole Hotel in room 911 and vomited in front of the elevator on the 9th floor. This led to the infection of vacationers and visitors on the 9th floor ,who then spread the SARS CoV to Hong Kong, Singapore, Hanoi, and Toronto. There was likely additional spread early, but the above four sites led to a significant number of cases in each location. SARS CoV had a high case fatality rate, especially in the older patients (40’s to 60’s), but spread was largely limited to the above locations, other than spread in mainland China and Taiwan.

In contrast, H1N1 rapidly spread worldwide and more than 90% of fatalities were in those under 65. Although the case fatality rate in the younger population is markedly higher than seasonal flu, the overall fatality rate is on a par with seasonal flu because most of those over 65 have immunity due to cross reactivity with the 1918 strain or older seasonal H1N1 sequences.

In Contagion, MEV had a very high case fatality rate (20-25%) and caused a massive number of fatalities (a bit too quickly) in the first month of spread. Thus, MEV had some aspects in common with SARS (geographic origin and high case fatality rate) and others in common with H1N1 (rapid worldwide spread). In both real cases the spread was reported promptly and sequences were generated and shared by the scientific community to determine the origin and evolutionary change via phylogenetic analysis, as was also seen in the movie, including a picture of the recombinant 3-D ribbon structure.

However, in addition to jumping from swine to human (as also seen for MEV), H1N1 has also jumped back to swine where there have been worldwide infections leading to additional evolution. This additional evolution has been seen in the recently released trH3N2 sequences, which have acquired the M gene from H1N1 via reassortment. This acquisition is in addition to changes acqruired via recombination, which includes the PB1 E618D in early human isolates, as well as several of the recent HA changes, which map to seasonal H3N2. Thus, trH3N2 is evolving via reassortment and recombination and detection of spread has only recently been released.

Thus, the effects of the H1N1 jump to humans has not been as lethal as MEV in initial cases, but H1N1 continues to spread, even after the development of a vaccine, and the influenza of H1N1 has now been seen in trH3N2 which will likely lead to another pandemic and co-circulation of two pandemic influenza’s leading to more recombination and more problems.

The influence of pandemic H1N1 and trH3N2 is still evolving and the potential for a significant impact remains, even after the development of a vaccine and widespread use of antivirals.

Thus, while H1N1 and H3N2 spread has many parallels with MEV spread and evolution as seen in “Contagion”, H1N1 and H3N2 are still evolving and the spread and effects are and will be quite real.

[dothedd]

CDC Media Myths On trH3N2 Sequence Relationships
Recombinomics Commentary 11:30
September 12, 2011


Q: What's the story behind a new swine flu virus that health officials are concerned about?

A: The federal Centers for Disease Control and Prevention reported Sept. 2 on two cases of children infected with new swine flu viruses. One child was in Pennsylvania; the other was in Indiana.

A few days later, Pennsylvania health officials reported that they had identified two additional children in that state with the new swine flu virus, and that all three children had attended an agricultural fair in southwestern Pennsylvania during the week of Aug. 13-20.

The Indiana case was not linked to the others, and the flu-virus strain was slightly different.

The above Q&A is from a media report published today, which reflects the comments by the CDC in the week 34 FluView as well as the early release MMWR which noted drift variations between the isolate in from an Indiana patient (2M), A/Indiana/08/2011 (2M), and the Schuylkill patient (2F), A/Pennsylvania/09/2011, who visited the Washington County fair on August 16 and provided samples during an emergency department visit on August 20.

However, the PA Department of Health subsequently reported two addition confirmed cases (both 9F) and samples collected on August 25 and August 26 generated sequences (A/Pennsylvania/11/2011 and A/Pennsylvania/10/2011, respectively) which matched the Indiana sequence and did not have the drift variation sequences seen in the Schuylkill patient. Thus, the constellation of the eight gene segments, including the M gene segment from pandemic H1N1 was the same for all four isolates, and the genetic drift was limited to the sequence from the Schuylkill isolate. The two Washington County isolates matched each other and the Indiana sequence.

The confusion created by the CDC report and failure to clarify after additional sequence data was generated from additional cases is similar to the confusion created by the WHO pager aler on trH3N2 in November, 2010. The alert cited isolates from two cases (one from Illinois which was represented by the sample described by Wisconsin, A/Wisconsin/12/2010, and the other from Pennsylvania, A/Pennsylvania/14/2010 isolated from a patient who developed symptoms 6 weeks after the Illinois patient. The two sequences were trH3N2 but distinct, especially in the NA gene, indocating they did not come from a common source.

However, in 2011 another Pennsylvania case, A/Pennsylvania/40/2010 was described, and that patient developed symptoms less than a week prior to the Illinois (Wisconsin) patient. Moreover, the sequences were virtually identical. Those cases were followed by a cluster from Minesota, and the sequences from the index case, A/Minnesota/11/2010, were virtually identical to the WI/12/10 and PA/40/10 sequences and five of the genes from those isolates (PB2, PA, HA, NP, NS) are in the recent 2011 isolates from Indianan and Pennsylvania.

Thus, in both cases the CDC cited differences between the common sequences and the exception, and ignored the identity between the dominant sequence identified in unrelated sequences and signaling human transmission.

These glaring omissions continue to increase pandemic concerns.

[dothedd]

CDC Media Myths On Swine Contact By trH3N2 Cases
Recombinomics Commentary 12:45
September 12, 2011


Flu viruses occasionally jump from pigs to people, but generally there isn't ongoing transmission; rather, each infected person has had some direct contact with swine.


The above comment is from a Q&A in a media report today, which propagates another media myth: trH3N2 cases are linked to direct contact with pigs. This myth is perpetuated by the CDC website on swine influenza in humans, and a CDC series of “Have you heard” reports for the media, which is used to perpetuate the myth. In reality most trH3N2 testing is limited to patients with some loose linkage to swine (live in the area where swine is present or attended an agricultural fair), who develop flu-like symptoms in the off season, such as three for the four recent trH3N2 cases.


Only one of the cases had reported contact with swine, a Schuylkill County resident (2F) in eastern Pennsylvania who visited the Washington County agricultural fair in western Pennsylvania on August 16. This case visited the Emergency Department on August 20 and after testing positive for influenza A, her sample was PCR tested for trH3N2. August 20 is during the off season for the northern hemisphere, so flu-like symptoms including breathing difficulties, coupled with a influenza A positive test and swine contact, led to the more specific test.

A more common scenario was reported for the case (2M) from Indiana, who did not have contact with swine. Instead, his caretaker did have swine contact, although neither the swine nor the caretaker had flu-like symptoms and none were confirmed to be infected with any influenza (as detailed in the early release MMWR). However, the swine linkage led to the trH3N2 test, not swine contact.

The same was true for the two Washington County cases, who attended the Washington County fair, but in contrast to the Schuylkill case, did not have swine contact. The sequences from the two Washington County cases matched the Indiana case, not the Schuylkill case, although all four were infected with the same constellation of genes.


Thus, all three cases who did not have swine contact had matching sequences, signaling human to human transmission, as was seen in the first two United States pandemic H1N1 cases reported in the April, 2009. Neither had contact with swine or each other, but were infected with the same pandemic H1N1 indicating undetected human cases were widely transmitting the virus.


Moreover, a recent report indicated the M gene segment was critical for the aerosol human to human contact driving the spread of pandemic H1N1 and the same M gene segment has been acquired by the four recent trH3N2 cases in Indiana and Pennsylvania.


Thus, human to human transmission of trH3N2 is supported by the lack of swine contact in most cases, sequence identity between the Pennsylvania and Indiana cases, the acquisition of the M gene from pandemic H1N1, and the clustering of sequences in human trH3N2 including cases from 2010 with an H3 (and additional internal genes) closely related to the sequences from the 2011 cases.

The media myths on trH3N2 cases and swine contact continue to raise pandemic concerns.

[dothedd]

Records Set By New trH3N2 Contagion Cluster
Recombinomics Commentary 11:30
September 12, 2011


The recent cluster of trH3N2 cases in Indiana and Pennsylvania has led to a number of CDC publications describing these cases, but the reporting has led to confusion in the media and the creation / propagation in a number of media myths. Although 8 confirmed cases of trH3N2 were reported for infections in 2009 (2 cases) and 2010 (6 cases), the cluster in 2011 has set new trH3N2 records in multiple categories and in triple reassortants in general if the 2009 H1N1 pandemic cases are excluded. In addition to the 12 trH3N2 cases, there were 13 trH1 cases (12 trH1N1 and 1 trH1N2) reported prior to the 2009 pandemic. Although all of the above are triple reassortants (tr), the pandemic H1N1 is readily distinguished from the 25 cases reported in the United States since 2005.

At the end of 2010 the CDC released sequences from the earlier tr cases, which indicated the human trH3N2 cases were clustering, although some similar swine isolates were reported. The clustering of the human cases, in spite of increased swine surveillance raised concerns that the trH3N2 cases were transmitting in humans.
These concerns were increased when the WHO issued a pager alert on two cases, a child from Illinois (later identified by Wisconsin as an infect boy with CDC sequence designated A/Wisconsin/12/2010) and an adult from Pennsylvania (A/Pennsylvania/14/2010). The alert created concerns, especially in eastern Europe, but concerns were reduced by statements indicating that the sequences were distinct and the trH3N2 was not spread in humans. However, a subsequent case, who represented another Pennsylvania infection, A/Pennsylvania/40/2010, was reported in 2011 because of technical issues in growing the virus, which had been designated seasonal H3N2 because the human H3 and human N2 had produced a positive result with sub-typing reagents. However, sequence data clearly showed that the virus was a trH3N2 and was closely related to WI/12/10. Since both cases were infected within a week of each other, the close genetic relationship was a concern. This concern increased when another case was identified in Minnesota, A/Minnesota/11/2010, was identified which was also closely related, and the daughter of the index case was serologically confirmed to have also been trH3N2 infected. This cluster led to the creation of a pandemic vaccine target, using the sequence from the index case.

However, the “records” set by the above cases were broken by the latest cluster, which were initially reported by the release of sequences from an Indiana sample collected July 24, 2011, A/Indiana/08/2011. The CDC released the sequence after a new case was added to the MMWR in early August, which was initially assumed to be the daughter in the Minnesota cluster. However, the July collection date of the Indian cas led to speculation that it had been the MMWR reported case, which remained unclear since the MMWR provided no detail.

However, when two more trH3N2 were reported in the week 34 MMWR, and one was from Indiana and the other from Pennsylvania, it became clear that 2nd 2011 case in the MMWR was another case from 2010, and the two cases reported in the same MMWR week were new and represented a record for reporting of trH3N2 cases, or triple reassortants in general, other than pandemic H1N1. This record was quickly followed by another “first” when the PA Department of Health and then CDC via a “have you heard” media update indicated that there were two more trH3N2 cases from Pennsylvania, raising to three the number of trH3N2 cases linked to a single event, the agricultural fair in Washington County.

In addition to representing the largest number of confirmed cases linked to a single event, the sequencing of the isolates set additional records. All four of the isolates from Indiana and Pennsylvania had acquired the M gene segment from pandemic H1N1 creating a new constellation of flu genes that had never been previously reported in humans or swine. Moreover, all of the trH3N2 genes also matched, and the sequences from the three cases with no swine contact were virtually identical creating the largest cluster of identical tr confirmed sequences since the pandemic H1N1 outbreak.

Moreover, the H3 sequences from these cases were related to the 2010 cluster, but had evolved further away from seasonal H3N2 sequences, so two of the sequences have been reported as unsubtypable, the first such designations since the H1N1 pandemic.

In addition to these “records” a recent report indicated that the jump of pandemic H1N1 from swine to humans was tightly associated with the M gene segment, which now was in the 2011 trH3N2 isolates increasing concerns that the “first” were due to the more efficient transmission of trH3N2 in humans, resulting in a new contagion.

In spite of these new “records” and alarming acquisition, CDC reports continue to note the similarities between the result isolates with earlier cases and downplay the significance of these recent events with statements from CDC officials as well as statements noting sequence differences between the Schuylkill isolate and the Indiana isolate and not commentating on the identity between the sequences from cases without swine contact.

Thus, the statements in the week 34 FluView, the week 34 MMWR, and the latest “have you heard” on these four cases have generated an number of media myths on the historical perspective and pandemic potential in the latest cluster including .sequence relationships and links to swine contact as well as the various recent records set.

Moreover, the request for samples from patients with flu symptoms and a swine connection adds to the confusion. Instead of limiting samples, the CDC should be collecting as many influenza A samples as possible and PCR testing these an earlier samples to get a true picture of the number of trH3N2 cases in the recent and current samples collected by the CDC and state departments of health.

Testing on a par with PCR testing for pandemic H1N1 is long overdue for H3N2 or influenza A positive cases.