China - H7N9 Human Isolates on Deposit at GISAID

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Ongoing Discussion of Hospital Over-Capacity Situations and UnExplained Pneumonia Cases in China contemporary to the disclosed Emergent H7N9 cases.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Remarks on

Host-Transition
Adaptation
in
Transport Vector Species

Ignition v. Spread

Re-published from an earlier thread with one of Al's wonderful maps and curiosity's insightful epidemiology, China - Map of H7N9 hot spots outside of Shanghai [FT #204004].

On another note: the term "hot spots" reminded me of the chronological onset and location map. IMO, the map looked similar to a few wildfire maps I've seen where the larger fire sends burning embers into the atmosphere that start smaller fires around it - often in the same general direction. Some of those surrounding fires light up, others smolder - dependent on fuel source, terrain, atmospheric conditions, etc...

China has a lot of fuel for "hot spots."

Although, depending on which 1918 origination scenario one thinks is most accurate - Haskell, Kansas; Asian source; or France source; etc... - fuel may or may not matter at the beginning.

Adaptation method and rate within the transport vector are the primary effectors at the beginning of a true influenza zoonotic. Reservoir dynamics are paramount to this process of avian-to-mammal bridging. The number and geographic dispersal of vector organisms (Passerines, Anser, Anas, et al) that are able to transport the host-switching virus describes the commonly found geographic outliers during similar times of ignition.

We all know that spread occurs in an arc, but spread is Stage 2, a separate process that begins only after a virus is established in a new host. Stage 1, Ignition, when traced carefully, may be found often as multi-focal and spontaneous.

Non-intuitive ignition may be seen in the recent GeneWurx prediction and validation of non-contiguous human H7N9 spontaneous emergence in land-locked Hunan province: Passerines Fly Higher than Poultry [FT#495364].

Based on a novel mechanism of genetic tracing that cross-references human clinical outcomes, calculations (guided by the recognition of a sub-segment antigen adaptation method) supported Ignition of emergent human cases in Guizhou and Hunan. We understood from the start that cases in Guizhou would not be reported. Non-contiguous emergent H7N9 (no geographic arc) for a human Hunan case was reported on the 27th of April with a case onset of 2013-04-14.

Human re-hosting will always occur when viral particles with the right receptor binding genetics are in proximity. That much is non-bypassable. If the new viral strain is successfully competitive over standing infective influenzas, then Ignition becomes pandemic.

But it all starts at the animal-man interface, at the contact vector that is also frequently the viral transportation. Outside of exceptional situations where innate immunity is improbably high or low in a vector-proximal, ignitable population, mankind as an effector (the fuel) is not so important.


Related Reading

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Mammalian Adaptation of Emergent H7N9 Neuraminidase in Jiangsu
• TamiFlu Resistance via NA 292K in Taiwan and Shanghai
• Zhejiang Fatal H7N9 with Divergent NA, PB2 701N and Aberrant PA
• Novel PB2, Divergent PB1 & Novel PA from First Fujian Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Remarks on

Re-Marketing
of
Toxic Anti-Virals

Treatment methods that are deemed successful by the practitioner must be vetted by detailed evaluation using statistically significant populations. We have learned that professed practical meaurements are not always the measure of good practice. Modalities based on low count measurements occuring in very transient and particular situations rarely translate to population-level benefits. We undertake an examination of basic facts concerning one treatment modality against emergent H7N9 influenza in pediatric patients.

Of course, we are quite concerned that emergent H7N9, if revised toward pandemic-enabling genetics, will have commensurately new attack groups, clinical presentations, treatment responses, clinical progressions, clinical outcomes and prognoses. What works today, or appears to work, may be ineffective or even dangerous against a revised dynamic.

With those items in mind, we re-publish comments from an earlier thread, Emerg Microbes Infect. Mild infection of a novel H7N9 avian influenza virus in children in Shanghai [Shanghai CDC: FT #503684].

Even in a crisis, some clinicians will exceed groupthink. If you refuse to rely on groupthink, please choose to review our categorical evaluation of this treatment modality.

[FT #503644]
It is also important to note the positive correlation between the influenza polymerase inhibitor ribavirin and the rapid clinical symptoms resolutions in the described cases. The PA inhibitory property is also cited in other paper cit.

Ribavirin is demonstrated to be profoundly toxic.

For 43 years.

Toxicity is a primary evaluation point around which the extensive body of ribavirin research revolves. Human studies, even of short duration, frequently result in subjects leaving the investigation prior to completion due to toxicity or other physiologically-limiting outcomes.

CardioVascular Contra-Indication

Furthermore, the known contra-indications of ribavirin are also common correlators to the subject groups and factors surrounding pandemic influenza or host-transition viral outbreaks. An obvious and prominent concern is that host-transition virus reservoirs dismay us with fatal clinical outcomes correlated with antecedent cardiac muscle failure, vascular musculature failure, disseminated intravascular coagulation (DIC) and organ failure (liver). The contra-indication profile of ribavirin provides direct risk factors in these areas. The thousands of amputations employed on children infected by pH1N1 after DIC allows a rational researcher to steer clear of ribavirin for future pandemic influenza.

Child-Bearing Age Contra-Indication

Citizens of Child-Bearing age (male and female) risk their future child's life and / or deleterious genetic mutation of their child when ribavirin is engaged. We are all familiar with the fact that host-transition viral reservoirs are dangerous to males and females of child-bearing age and that pregnancy creates exceptional risk of high morbidity and fatality with host-transition viral reservoirs. Ribavirin as a standard in an influenza pandemic risks damaging the current and the future generation.

Eye Concerns

Additionally, retinopathy is associated with the more modern combinational therapies employing this toxic pharmaceutical. As we have recently seen, H7 influenza serotypes tend toward ocular involvement. Human H7N7 outbreaks showed conjunctivitis as a common presenting symptom. Emergent H7N9 infection through the eye is demonstrated in the lab to result in mammal respiratory infection [US CDC: FT#503653]. Maintaining eye integrity may feature as a public health recommendation during an H7 pandemic.

Immuno-Suppression

Ribavirin and other inhibitors in the IMPDH group are recognised for immuno-suppressive activity and are often employed for that particular mechanism of action. Like many influenza reservoirs (pH1N1) and individual clades, emergent H7N9 is proposed, even by the authors of this paper, to be microbiologically and clinically immuno-suppressive.

Unintended amplification of immuno-suppression is a risk that may lead to super-infection when treating an emerging pandemic virus with ribavirin. Super-infection, we are told, is a substantial contributor to fatality in pandemic influenza. Considering the extended durations in hospital observed with the first wave of emergent H7N9 human cases, clinicians will be wise to consider the potential gravity of amplified immuno-suppression leading to super-infection and then fatality.

Emerging Microbes & Infections (2013) 2, e41; doi:10.1038/emi.2013.41 - Published online 10 July 2013

Therefore, we propose that the novel H7N9 virus may induce transient immunosuppression that occasionally results in fatal opportunistic infections, especially in patients with underlying diseases.

Revenue Enhancement

Re-Marketing has become a revenue enhancement strategy promoted and economically idealised by medical consultants over the last decade. The failure of corporations to bring us working solutions provides those very same teams the suffering subjects they need to re-market their previous bad ideas (because nothing new is available). This economically beneficial re-cycling of inappropriate and out-dated techniques has recently resulted in medical tragedies.

While we may hold some sympathy for the frustrated clinicians who are influenced by these advising vultures to "throw everything you have at that infection", we also hold those clinicians to the responsibility of providing some semblence of input toward THEIR patient's treatment. Due to the vacuum found where new ideas should be arising, off-label usage is too regularly adopted by leading professors and clinicians for untested, even bizarre, treatment modalities.

Ribavirin as a standard for influenza care surpasses bizarre, however, and moves into the patently absurd. As if the blind being allowed to lead the blind is no longer enough, we are now being asked to allow the blind to create the blind?

Originally published:
Researcher devises drug cocktail for coronavirus [FT Thread #493730]

A primary issue here is that ribivarin - whilst an effective anti-viral agent - is highly toxic. For this reason it was largely discounted as potential treatment for H5N1 human infections.

See http://www.drugs.com/pro/ribavirin.html

The primary clinical toxicity of Ribavirin is hemolytic anemia. The anemia associated with Ribavirin therapy may result in worsening of cardiac disease and lead to fatal and nonfatal myocardial infarctions. Patients with a history of significant or unstable cardiac disease should not be treated with Ribavirin [see Warnings and Precautions (5.2), Adverse Reactions (6.1), and Dosage and Administration (2.3)].

Significant teratogenic and/or embryocidal effects have been demonstrated in all animal species exposed to Ribavirin. In addition, Ribavirin has a multiple dose half-life of 12 days, and it may persist in non-plasma compartments for as long as 6 months. Therefore, Ribavirin, including Ribavirin tablets, is contraindicated in women who are pregnant and in the male partners of women who are pregnant. Extreme care must be taken to avoid pregnancy during therapy and for 6 months after completion of therapy in both female patients and in female partners of male patients who are taking Ribavirin therapy. At least two reliable forms of effective contraception must be utilized during treatment and during the 6 month post treatment follow-up period

Related Reading

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Mammalian Adaptation of Emergent H7N9 Neuraminidase in Jiangsu
• TamiFlu Resistance via NA 292K in Taiwan and Shanghai
• Zhejiang Fatal H7N9 with Divergent NA, PB2 701N and Aberrant PA
• Novel PB2, Divergent PB1 & Novel PA from First Fujian Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Scalability Questions
Around
Rate of Mild Case Detection

RePublished from BMJ. Detection of mild to moderate influenza A/H7N9 infection by China?s national sentinel surveillance system for influenza-like illness: case series [Thread#207942]:

Post#3
The number of assumptions is skyrocketing as the number of possible cases. And this is not much informative.

554 sentinel points
12.5 swabs per week / point
554*12.5*4 wk=27,700 swabs per month

5 sentinel swabs tested positive for H7N9 from the above paper =

5 / 27,700 = 0.02% positivity rate

If in a 5,000,000 China megacity during the same period of time there was an ILI rate of 14 x 1,000 then:

14 : 1,000 = x : 5,000,000

x = 70,000 ILI cases per week

if these ILI cases should have been swabbed - employing the above positivity rate we should have had:

70,000 * 0.02% = 14 H7N9 cases

Scaling

Scaling statistics using national factors onto discrete (sub-component) populations is not generally held as an ideal mechanism for creating actionable results at the discrete population level. When this type of spreading is undertaken at a giga-scale (i.e. population of China), often additional considerations and cautions must be employed. The types of projections are the purest form of invention.

An inspection of significant digits may be in order for these calculations while keeping at the forefront the fact that averages and assumptions are being multiplied by averages and assumptions. We will examine here the individual factors and the calculation veracity. Projection estimates will be provided at the end of this post for your example population of 5 million and for several actual geographies in China related to emergent H7N9.

You certainly are accurate on the skyrocketing effect of assumptions. One paper that you posted requires a minimum of 15 swabs per week and this paper suggests a quota range of 10 to 15 swabs per week, a ceiling 50% above the floor (substantial variance, ? sqared=6.25). Using actual counts from the earlier study (46,807 swabs from 554 locations over 8 weeks) gives an average of 10.56115 swabs per location per week.

Factor Validity

The authors openly admit that swabbing is not random and then they immediately attempt without any observational evidence to attach the idea that this quota-driven, non-random test is unbiased? They pledge that their system, though structured with a non-randomised collection, is representative of a billion count population? The system begins by missing 97.5% of the hospitals (sampling 2.5%) and then proceeds to require 15 swabs per week, but only gets about 10.56115 across a week's duration. We're not certain if the authors have supplied a supplement denoting the actual geographic collections? If an attempt is being made to differentiate disease epidemiology, then we'll need specific geographic counts rather than obtuse gerrymandering accumulations and averages.

Severity bias is generally present in infectious disease collections that are not intentionally randomised.

Test Duration

Is the test duration for this paper defined or are we to assume that the accumulations take place over the entire emergence period from their first publicly reported case in late February until May 27? If so, then 12 weeks very much dilutes the outcome when used in place of the 4 week estimate to accommodate the 5 mild cases discovered via passive surveillance of ILI reports. Early multipliers reduced by 66.67% dampen results.

Emerg Infect Dis. Monitoring Avian Influenza A(H7N9) Virus through National Influenza-like Illness Surveillance, China [Thread #205425, Post#1] (emphasis mine)

On April 3, 2013, to enhance surveillance for influenza A(H7N9) virus, all network laboratories were required to increase the number of specimens to a minimum of 15/week and to test all specimens collected since March 4, 2013, for influenza A(H7N9) virus by real-time reverse transcription PCR . . .
. . .
During March 4?April 28, CNISN tested 46,807 nasopharyngeal swab samples from 554 sentinel hospitals throughout mainland China.

ReCalculation

Information Quality is very low in the emergent H7N9 discussion though Information Quantity sometimes exceeds necessity.

Using the actual numbers from the previous study (46,807 covering 8 weeks from early March to late April) summed with the mandated 15 swabs per week per hospital (33,240 covering 4 weeks from late April to late May), we estimate the denominator to be a minimum of 80,047 total swabs over 12 weeks. With detection of 5 mild cases, the rate calculates to:

Mild Case Detection Rate for Emergent H7N9 in ILI Cases ~ 0.006246329%.

We carry forward without examination the Influenza-Like-Illness basis from the nominitive Michieli proposal. Holding constant the discussed ILI factor of 0.01400 (14 Reports per 1,000 Population), the following selected geographic projections may be discussed based on the limitations of the incoming factors' veracity. Particularly daunting is the non-applicability of a national Mild Case Detection Rate across disparate, sub-state geographies.

Concentrated City
Population . . . . . 5,000,000
Projected ILI . . . . . .70,000
Projected Mild H7N9 Count___4.4

Beijing
Population . . . . 20,690,000
Projected ILI . . . . .289,660
Projected Mild H7N9 Count__18.1

Shanghai
Population . . . . 23,470,000
Projected ILI . . . . .328,580
Projected Mild H7N9 Count__20.5

Guizhou Province
Population . . . . 34,690,000
Projected ILI . . . . .485,660
Projected Mild H7N9 Count__30.3

Fujian Province
Population . . . . 37,200,000
Projected ILI . . . . .520,800
Projected Mild H7N9 Count__32.5

Zhejiang Province
Population . . . . 54,630,000
Projected ILI . . . . .764,820
Projected Mild H7N9 Count__47.8

Jiangsu Province
Population . . . . 78,990,000
Projected ILI . . . 1,105,860
Projected Mild H7N9 Count__69.1

China
Population . . 1,344,000,000
Projected ILI . . .18,816,000
Projected Mild H7N9 Count__1,175.3

As Chris & Edgar like to say, "Caveat Lector."

Genetic Calibration

We do tend to agree with the authors that cross-referencing the actual disease (gene form) to the statistics is potentially beneficial for the public.

Related Reading

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Mammalian Adaptation of Emergent H7N9 Neuraminidase in Jiangsu
• TamiFlu Resistance via NA 292K in Taiwan and Shanghai
• Zhejiang Fatal H7N9 with Divergent NA, PB2 701N and Aberrant PA
• Novel PB2, Divergent PB1 & Novel PA from First Fujian Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[sharon sanders]

Re: China - H7N9 Human Isolates on Deposit at GISAID

bump this.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

GISAID Citations

Novel PB2, Divergent PB1 & Novel PA
from
First Fujian Case

We would like to thank the researchers at the Fujian Center for Disease Control and Prevention for their Emergent H7N9 supplementary GISAID deposit on 2013-06-04 dated 2013-05-27. The Polymerase and NP segments were deposited under a separate isolate identifier than the earlier HA, NA, MP and NS deposit leaving questions concerning passage history for the first GISAID isolate identifier (140881). No metadata or clinical outcome was provided by the depositors, although the FluTrackers 2013 Human Case List [#116] indicates an onset of April 18 and hospitalisation at April 23. The patient was discharged on May 13. This 65 year old male subject was sampled on April 24, 2013 and passage was annotated as 'E1'.

The case follows course having PB2 627K and a stock emergent H7N9 Hemagglutinin segment, but was originally made novel by the NA 148E (147E). The Neuraminidase polymorphism is found on the Indonesian H5N1 human Karo cluster from May 2006. The Fujian emergent H7N9 HA and NA were discussed on May 9, 2013 at FluTrackers post, Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case, [#107] within this thread.

This divergent Polymerase Basic 1 segment carries 7 polymorphisms, 5 that are not previously found on emergent H7N9. However, the bracketing polymorphisms, PB1 56A and syn544A (GCc), are previously found within ΣeH7N9.

The Polymerase complex segments (PB2, PB1, PA) were referenced against their respective ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f segments.

PB2 Polymorphisms

. . . . ChinaFujianLongyanCity1_E1_65M_2013_04_24_s (
. . . . . . . . GISAID PB2 EPI453827
. . . . . . . . GISAID Isolate EPI_ISL_142319
. . . . . . . . 2 Polymorphisms (1 Amino and 1 Silent)
. . . . . . . . syn244Y (TAt) [eH7N9 ckChinaZhejiangDTIDZJU01_2013_04,
. . . . . . . . . . . . . . . . . . . . . . . . envChinaHangzhou34_2013_04_04],
. . . . . . . . 420L [H7N9 Novel])


PB1 Polymorphisms

. . . . ChinaFujianLongyanCity1_E1_65M_2013_04_24_s (
. . . . . . . . GISAID PB1 EPI453828
. . . . . . . . GISAID Isolate EPI_ISL_142319
. . . . . . . . 7 Polymorphisms (5 Amino and 2 Silent)
. . . . . . . . PB1 Truncated before aa2,
. . . . . . . . 56A [eH7N9 pgnChinaShanghaiS1069_2013_04_02],
. . . . . . . . syn64P (CCt) [H5N1 China Human Fatality Cluster February 2013 with HA 202V:
. . . . . . . . . . . . . . . . . . . . . . . [EPI420390] ChinaGuizhou1_E1_21F_2013_02_08_f,
. . . . . . . . . . . . . . . . . . . . . . . [EPI420767] ChinaGuizhou2_E1E1_31M_2013_02_09_f],
. . . . . . . . . . . . . . . . . . [H5N1 China Hunan environment Dongting Lake 2008],
. . . . . . . . . . . . . . . . . . [H5N1 Bangladesh Avian gal 2012],
. . . . . . . . . . . . . . . . . . [H5N1 America Avian anas 2010],
. . . . . . . . . . . . . . . . . . [H5N1 India Avian gal 2010],
. . . . . . . . . . . . . . . . . . [H5N1 India Avian pas 2008],
. . . . . . . . . . . . . . . . . . [H9N2 China Avian gal 2011],
. . . . . . . . . . . . . . . . . . [neH7N9 Avian Alaska & Korea],
. . . . . . . . . . . . . . . . . . [H7N7 America Avian anas 2010],
. . . . . . . . . . . . . . . . . . [H7N7 Korea Avian anas 2009],
. . . . . . . . . . . . . . . . . . [H7N7 Korea Avian pas 2007],
. . . . . . . . . . . . . . . . . . [H7N7 Australia Avian pas 1985],
. . . . . . . . . . . . . . . . . . [H7N6 Japan Avian quail 2009],
. . . . . . . . . . . . . . . . . . [H3N2 China Avian anas 2011],
. . . . . . . . . . . . . . . . . . [H11N9 China Jiangxi Avian environment 2009],
. . . . . . . . . . . . . . . . . . [H11N9 America Avian anas 2009],
. . . . . . . . 338G [H7N9 Novel],
. . . . . . . . 373P [H7N9 Novel],
. . . . . . . . 376V [H7N9 Novel],
. . . . . . . . 377G [H7N9 Novel],
. . . . . . . . syn544A (GCc) [H7N9 Widespread (18)],
. . . . . . . . . . . . . . . . . . . [H5N1 China Human Fatality Cluster February 2013 with HA 202V:
. . . . . . . . . . . . . . . . . . . . . . . . [EPI420390] ChinaGuizhou1_E1_21F_2013_02_08_f,
. . . . . . . . . . . . . . . . . . . . . . . . [EPI420767] ChinaGuizhou2_E1E1_31M_2013_02_09_f])

PA Polymorphisms

. . . . ChinaFujianLongyanCity1_E1_65M_2013_04_24_s (
. . . . . . . . GISAID PA EPI453837
. . . . . . . . GISAID Isolate EPI_ISL_142319
. . . . . . . . 3 Polymorphisms (2 Amino and 1 Silent)
. . . . . . . . syn58G (GGc),
. . . . . . . . 201T [eH7N9 Novel],
. . . . . . . . . . . . [neH7N9 dk_w_spotbillKorea447_2011_04],
. . . . . . . . 403V [H7N9 Novel])

H7N9 Influenza PolymeraseBasic2, PolymeraseBasic1 and PolymeraseAcidic Segments elucidated at 2013-06-04-03_11_36_299990 by GeneWurx see.PolyDetector v0, Copyright 2007-2013

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & from GISAID’s EpiFlu™ Database on which this research is based. An additional list is detailed in the linked PDF entitled "GISAID_Citations_H5N1_2011" at Is H7N9 Spreading from Human to Human in China? Post#164

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

GISAID Citations

Zhejiang Fatal H7N9
with
Divergent NA, PB2 701N and Aberrant PA

We would like to thank the researchers at Zhejiang University for their GISAID deposit on 2013-04-17 that included this divergent passaging of one emergent H7N9 fatal human sequence. The Hemagglutinin and Polymerase Basic 1 are stock emergent H7N9, ordinary. However, the Polymerase Acidic segment carries 36 polymorphisms with striking Human H5N1 sub-segment homology to:

• China Wet Market 2009 ~23 polymorphisms
• Vietnam Fatality 2008 . ~21 polymorphisms

The Polymerase complex segments (PB2, PB1, PA) were referenced against their respective ChinaAnhuiChuzhouCity1_E1_35F_2013_03_20_f segments, the Hemagglutinin and Neuraminidase against a pseudo-consensus.

PB2 Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID PB2 EPI441801
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 3 Polymorphisms (3 Amino and 0 Silent)
. . . . . . . . 395S [ChinaShanghai1_E1_87M_2013_03_04_f],
. . . . . . . . 627E [ChinaZhejiang2_64M_2013_04_03_f,
. . . . . . . . . . . . . . Nanjing1_45F_2013_03_28,
. . . . . . . . . . . . . . TaiwanTaipeiT02081_53M_2013_04_22_s],
. . . . . . . . . . . . . . [H7N9 Avian],
. . . . . . . . 701N [ChinaZhejiang2_64M_2013_04_03_f])

PB1 Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID PB1 EPI441800
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 1 Polymorphisms (0 Amino and 1 Silent)
. . . . . . . . syn544A (GCc))

PA Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID PA EPI441795
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 36 Polymorphisms (5 Amino and 31 Silent)
. . . . . . . . . . . . . . ~ H5N1 Human China Wet Market 2009 (23 polymorphisms)
. . . . . . . . . . . . . . ~ H5N1 Human Vietnam Fatality 2008 (21 polymorphisms)
. . . . . . . . syn7Q (CAa) [H5N1 Human China, Vietnam],
. . . . . . . . syn58G (GGc) [H5N1 Human China, Vietnam],
. . . . . . . . syn62I (ATt) [H5N1 Human China, Vietnam],
. . . . . . . . 70V,
. . . . . . . . syn98T (ACg),
. . . . . . . . 100V [H5N1 Human China, Vietnam],
. . . . . . . . syn116R (CGa) [H5N1 Human China, Vietnam],
. . . . . . . . syn136N (AAc) [H5N1 Human China],
. . . . . . . . syn146H (CAc) [H5N1 Human China, Vietnam],
. . . . . . . . syn148F (TTc) [H5N1 Human China, Vietnam],
. . . . . . . . syn203E (GAg),
. . . . . . . . syn248Q (CAa) [H5N1 Human China, Vietnam],
. . . . . . . . syn257I (ATc),
. . . . . . . . syn262R (AGg) [H5N1 Human China, Vietnam (3rd Base)],
. . . . . . . . 272D [H5N1 Human China, Vietnam],
. . . . . . . . syn318K (AAg),
. . . . . . . . syn390L (cTG) [ChinaShanghai1_E1_87M_2013_03_04_f],
. . . . . . . . . . . . . . . . . . [H5N1 Human China, Vietnam],
. . . . . . . . 394D [H5N1 Human China, Vietnam],
. . . . . . . . 409S [H5N1 Human China, Vietnam (2nd Base)],
. . . . . . . . syn414A (GCa) [H5N1 Human China, Vietnam],
. . . . . . . . syn417L (tTG) [H5N1 Human China, Vietnam],
. . . . . . . . syn419D (GAt) [H5N1 Human China, Vietnam],
. . . . . . . . syn433A (GCt) [H5N1 Human China, Vietnam],
. . . . . . . . syn446F (TTc) [H5N1 Human China, Vietnam],
. . . . . . . . syn483I (ATc),
. . . . . . . . syn489C (TGc) [H5N1 Human China, Vietnam],
. . . . . . . . syn500L (CTa),
. . . . . . . . syn508R (AGg),
. . . . . . . . syn510H (CAc) [H5N1 Human China],
. . . . . . . . syn558S (TCt),
. . . . . . . . syn609K (AAg) [ChinaShanghai4664T_2013_03_05],
. . . . . . . . syn616S (TCt),
. . . . . . . . syn623E (GAa) [H5N1 Human China, Vietnam],
. . . . . . . . syn625P (CCc),
. . . . . . . . syn648S (AGt),
. . . . . . . . syn681F (TTc) [H5N1 Human China, Vietnam])

HA Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID HA EPI441794
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 104 Polymorphisms (17 Amino and 87 Silent)
. . . . . . . . 11I [#4I],
. . . . . . . . 130A [118A],
. . . . . . . . 183S [174S],
. . . . . . . . 188V [179V],
. . . . . . . . 195V [186V],
. . . . . . . . 198A [189A],
. . . . . . . . 211V [202V],
. . . . . . . . 217N [208N],
. . . . . . . . 235L [226L],
. . . . . . . . 285N [277N],
. . . . . . . . 307D [299D],
. . . . . . . . 321R [313R],
. . . . . . . . 410N [401N],
. . . . . . . . 427I [418I],
. . . . . . . . 455D [446D],
. . . . . . . . 462K [453K],
. . . . . . . . 541V [533V])

NA Polymorphisms

. . . . ChinaZhejiangDTIDZJU01_64M_2013_04_03_f (
. . . . . . . . GISAID NA EPI441797
. . . . . . . . GISAID Isolate EPI_ISL_139364
. . . . . . . . Deposited 2013-04-17 by Zhejiang University
. . . . . . . . 35 Polymorphisms (13 Amino and 22 Silent)
. . . . . . . . 16I [16I],
. . . . . . . . . . . [H5N1 Human Fatality China 2011],
. . . . . . . . . . . [H5N1 Human Cambodia 2005],
. . . . . . . . . . . [H5N1 Avian Rare (58) Scotland, Middle East and Asia]
. . . . . . . . . . . [pH1N1 Rare (35) Worldwide including Scotland and 1 US Low Reactor],
. . . . . . . . . . . [avH1N1farm],
. . . . . . . . 19A [19A],
. . . . . . . . . . . [avH1N1, H9N2],
. . . . . . . . 40G [40G],
. . . . . . . . . . . [H9N2],
. . . . . . . . 53T [53T],
. . . . . . . . 81T [80T],
. . . . . . . . . . . [avH1N1],
. . . . . . . . 84N [83N],
. . . . . . . . 112S [111S],
. . . . . . . . . . . . [H9N2],
. . . . . . . . 335I [332I],
. . . . . . . . . . . .[H6N1],
. . . . . . . . 359A [355A],
. . . . . . . . 401A [397A]
. . . . . . . . . . . . [H6N1],
. . . . . . . . 439L [437L],
. . . . . . . . 442G [439G],
. . . . . . . . . . . . . . [Emergent H7N9 ckZhejiangDTIDZJU01_2013_04],
. . . . . . . . 443D [440D])

H7N9 Influenza Hemagglutinin and Neuraminidase Segments elucidated at 2013-04-23-00_30_18_809650 by GeneWurx see.PolyDetector v0, Copyright 2007-2013
H7N9 Influenza PolymeraseBasic2, PolymeraseBasic1 and PolymeraseAcidic Segments elucidated at 2013-06-02-02_04_58_819480 by GeneWurx see.PolyDetector v0, Copyright 2007-2013

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

TamiFlu Resistance
via
NA 292K

www.thelancet.com Published online May 28, 2013 http://dx.doi.org/10.1016/S0140-6736(13)61125-3
Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

Four of the fourteen Sanger Neuraminidase sequences deposited at GenBank by Fudan University today and associated with the 2013-05-28 AntiViral Resistance paper (Malik Peiris and associates, The Lancet) show NA 292K. Two patients were indicated in the paper as developing antiviral resistance at NA 292K.

We are grateful to Dr. Peiris for coordinating production of the cross-reference information with the authoring organisations concerning serialisation and patient assignment for the samplings.

NA 292K

Our preliminary groupings for the four genotypic AntiViral Resistant sequences are estimated until we receive the master cross-reference. These groups represent either patient 2 or patient 3 from the tables in the paper. ECMO was required for each. Patient 2 expired at 19 days post-onset; patient 3 remained under treatment at 46 days (as of May 18, 2013). Group 1 is very likely Patient 2 who died during ECMO treatment. At least one fatality is represented in these 2 groupings.

We have arranged Group 1 due to Hemagglutinin homology:

Group 1 with 3 Sequences
Probable as Patient 2 Fatality
Drug Resistance by Treatment Day 4

• ChinaShanghai5240T_88M_2013_04_25_TmX_f [KF028381]
• ChinaShanghai5180T_88M_2013_04_23_TmX_f [KF028380]
• ChinaShanghai5083T_88M_2013_04_20_TmX_f [KF028379]

Group 2 with 1 Sequence
Probable as Patient 3

• ChinaShanghai4842T_56M_2013_04_13_TmX_s [KF028383]

NA Polymorphism Summary

ChinaShanghai4798T_62M_2013_04_12_TmX_s [KF028387] shows 152K and is likely Patient 6 who was mechanically ventilated and successfully discharged 35 days from onset. The polymorphism developed after day 3 of oseltamivir treatment and by the sampling date on day 5 of treatment.

ChinaShanghai4821T_2013_04_12_s [KF028392] shows NA 285K [284K] with first and third base polymorphisms. Only the emergent H7N9 ChinaShanghai4664T_2013_03_05 [KC853231] and a single instance from H4N9 avian show this polymorphism in the data record.

HA Polymorphism Summary

Three HA sequences carry a synonymous change at the third base of aa471 producing syn471E (GAg) [syn462E (GAg)]:

Probable as Patient 2 Fatality

• ChinaShanghai5240T_88M_2013_04_25_TmX_f [KF028376]
• ChinaShanghai5180T_88M_2013_04_23_TmX_f [KF028375]
• ChinaShanghai5083T_88M_2013_04_20_TmX_f [KF028374]

All three are also TamiFlu Resistant and are from a patient that required ECMO.

This syn462E (GAg) polymorphism has been seen in non-emergent H7N9 and in 5 emergent Human H7N9 sequences, including 3 from the single Taiwan case and 2 from the Pasteur Shanghai GISAID deposit of preliminary sequences on 2013-05-23.

The revision is also found in Pandemic H1N1 2009 (pH1N1), H5N1 (wildtype), H7N3 (extensive), H7N7 (rare, passerine), H9N2 (extensive, ostrich) and H9N9. Four of the nine instances within pH1N1 (all host transition from 2009) are from China and three others are from neighboring Asian countries. Argentina shows 2 sequences from subjects aged 2 years and younger. Seven of the eight pH1N1 sequences with this polymorphism and age metadata are pediatric cases and one of those Chinese sequences was published as part of a drug resistance investigation.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

From Tamiflu-Resistance Gene in H7N9 Bird Flu Spurs Drug Tests thread:

Almost all patients responded to oseltamivir treatment.

In some ECMO patients, and / or under corticosteroids treatment, R292K mutation in the NA happened, despite initial viral suppression.

During the course of treatment, a viral load 'rebound' was observed and it was suggested that either the immune status of patient or the concurrent use of steroids made the emersion of R292K much easy than in other patients.

Mixed population of susceptible and resistant to oseltamivir H7N9 viruses in certain cases has been noted from ALL the WHO updates so far.

Since the old age of the patients and the severity of the disease in these cases, this represents an expected finding as happened for H5N1 cases in the past.

Functional antivirals tests performed on R292K isolates demonstrated they are susceptible to oseltamivir and zanamivir albeit with some degree of variability.

It should be used a combination therapy in order to safeguard the limited arsenal of antivirals currently available for treatment and for pandemic preparedness stockpile.

An expedited clinical testing of such combinations should be performed as soon as possible in addition to an enhance surveillance for resistant strain in human and animals.

GM

Almost all Influenza cases self-resolve without treatment of any kind (~99.1 to 99.4%).

We can hardly nominate this situation as a clinical success when a substance designed to prevent the release of viral RNA from human cells preceded a finding of viral RNA in the blood from 86% (12 of 14) of professionally-treated patients. As you may recall, virus in the blood is a rarity in human influenza infections and is a direct indicator of increased morbidity. The entire cohort of 14 patients was treated aggressively with oseltamivir.

A 100 fold reduction in inhibition due to the NA 292K is no minor matter when 5-fold increase in IC50 is a traditional boundary for gathering statistics. Drug resistance was recorded as early as 5 days after treatment initiation.

Table 1: Demographic details, therapy, and outcome of patients with A/H7N9 infection appears to indicate that multi-drug antiviral therapy contributed at this time to only 1 successful discharge (Patient 8 at 22 days) with 1 remaining under standard treatment (Patient 5 at 37 days) and with 1 remaining on ECMO (Patient 3 at 46 days) at the time of data gathering. One death has also occurred after multi-drug treatment (Patient 2 at 19 days).

Ergo, at the time of data gathering, the "Released versus Deceased" counts are equal for this cohort (1/1) . . . demonstrating something much less than a stellar summary.

Despite the continual applause at these self-declared victories, the actual data predicts a future catastrophic clinical failing (even where logistics allow heroic measures) should emergent H7N9 transmission increase . . .

We would like to see an emergence of transparency in China by their reporting all of the current cases before the world is faced with a simultaneous emergence of H7N9 pathogen on all populated continents.

Their publication concerning the Beijing case is a start. Now let's put all the data on the table so that strategies may be developed that show potential for success.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Fatalities
Associated With
TamiFlu Resistance
via
NA 292K

Of the 5 sequences representing 4 human cases that carry the NA 292K polymorphism, 2 fatalities have occurred, both in Shanghai. At least 3 of the 4 cases required treatment involving in extremis measures such as ECMO.

Today's publication in The Lancet from Dr. Malik Peiris and an extensive multi-disciplinary team of Chinese organisations defines clinical progressions, treatments, outcomes and experimental findings including discussions on two TamiFlu Resistance markers, NA 292K and NA 152K. Their initial study reveals that the single 292K polymorphism increases oseltamivir resistance 100 fold.

www.thelancet.com Published online May 28, 2013 http://dx.doi.org/10.1016/S0140-6736(13)61125-3
Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

We have shown that A/Shanghai/1/2013 (H7N9) virus isolate contains a mixed population of Arg/Lys at position 292 of the NA gene, and by purifying virus plaques that carry NA Arg292 and Lys292, we have noted that this mutation increases resistance to oseltamivir by 100-fold and zanamivir by 30-fold in a fluorescence-based NA inhibition assay (unpublished data).

In patient 6 (from the mechanical ventilation group), we found emergence of Arg152Lys mutation in the NA gene. This mutation was first reported from an
immunocompromised patient infected with influenza B after zanamivir treatment.13 Using the baculovirus expressed N9 NA protein, it had been shown to exhibit mild resistance to both zanamivir and oseltamivir in vitro.14

We have been concerned that the few detailed clinical progressions may not have fully reported sepsis. This paper provides insight confirming one type of clinical similarity to other zoonotics including many pH1N1 cases and H5N1 human infections:

We detected viral RNA in the serum obtained at some time during the clinical illness of 12 (86%) of the 14 patients (appendix; all three patients in the ECMO group, all four patients in the mechanical ventilation group, and five of seven patients in the pneumonia group).

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

TamiFlu Resistance
via
NA 292K
in
Taiwan and Shanghai

For the sake of clarity, these findings were also cited in the recent WHO/China Joint Mission assessment paper: http://www.who.int/influenza/human_a...eport2013u.pdf


(...)

The first three isolates contained a number of genetic signatures previously associated in other subtypes with low pathogenicity in poultry, enhanced capacity for mammalian infection, resistance to the adamantane class of antiviral drugs and sensitivity to the neuraminidase inhibitors oseltamivir and zanamivir. These signatures include the following:

• A single arginine at the HA cleavage site, consistent with low pathogenicity in poultry;
• Deletion of five amino acids in the NA stalk, associated with adaptation to poultry;
• Q226L substitution in the HA, associated with enhanced binding to the α-2,6-linked sialylated receptors found in the mammalian respiratory tract;
• E627K in the PB2 protein, associated with viral replication at the lower temperature of the mammalian respiratory tract;
• S31N in the M2 protein, conferring resistance to adamantanes;
• Absence of the H275Y substitution in the NA, associated with resistance to the oseltamivir in H1N1 viruses;
• R292K in one virus (A/Shanghai/1/2013), associated with markedly reduced sensitivity to oseltamivir and modestly reduced sensitivity to zanamivir.

In vitro analyses confirmed that all three viruses bound both α -2,3- and α -2,6 linked sialic acids, suggesting an ability to bind to both avian and mammalian cells.

This analysis was published first at 2013-05-27-08:43 on an earlier thread entitled Drug-resistant H7N9 strains to change treatment: researcher as an up-to-the-minute TamiFlu Resistance summary of emergent ΣH7N9 in reply to Giuseppe's excellent synoptic of the press release. Sequences found only at GenBank, only at GISAID and at both were aggregated into a superset, de-duplicated, transformed and evaluated for this analysis:

The original deposit of the Taiwan E1 passage does not show the TamiFlu Resistance marker, but 2 isolates of ostensibly (no metadata) alternate tissue samples from a subsequent Taiwan deposit show the same change as the emergent H7N9 index case from Shanghai and two other May samplings from the Pasteur Shanghai release.

At this time, 5 of the emergent H7N9 human sequences indicate genotypic TamiFlu Resistance.

Another researcher today published suspiciously parallel, but un-attributed, material approximately 2 hours later at 10:41 AM. This pattern of a 15 minute to 2 hour reproduction cycle has drawn our focus and is observed weekly. The re-publication this morning unfortunately misrepresents the molecular mechanism. When a researcher cannot be troubled to conduct original research, then a direct quote with attribution is in order to prevent misrepresentation.

That individual appears to be interchanging this particular NA R292K (N2 numbering) genotypic marker with the amino acid position 294 (N2 numbering) polymorphism, NA N294S, that is also associated with TamiFlu Resistance on other backgrounds. Aoki, Boivin and Rogers discussed the molecular mechanism for 292K in Antiviral Therapy 12:603-616, 2007, Influenza virus susceptibility and resistance to oseltamivir [pdf] as defined by Kati, et al in Antimicrobial Agents and Chemotherapy, 2002, In Vitro Characterization of A-315675, a Highly Potent Inhibitor of A and B Strain Influenza Virus Neuraminidases and Influenza Virus Replication [pdf].

We would like to clarify specifically that the emergent H7N9 TamiFlu Resistance is being discussed due to the mechanism at amino acid position 292 (N2 numbering) as defined in the Aoki, et al review, though the polymorphism is found in absolute ordination on the H7N9 at aa294 of the Neuraminidase sequences. In order to support Cross Serotype Analysis, the polymorphism is also carried alternately as NA 293K in GeneWurx's present modeling instantiation.

Additionally, we formally declare that GeneWurx RnR Analytic reporting on emergent H7N9 stands separate from those who re-publish our findings without attribution and with insertional errors.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Mammalian Adaptation
of
Emergent H7N9
Neuraminidase

A human sequence, ChinaNanjing1_2013_03_28 [KC896776], was placed on deposit at GenBank by the Jiangsu CDC on 2013-05-23. Additionally, on 2013-05-26 the Zhejiang University School of Medicine deposited a human sequence, ChinaZhejiangHZ1_2013_04 [KF055467], also at GenBank with similar Neuraminidase findings. We estimate that the Zhejiang sequence is from Hangzhou due to name encoding (HZ) and sequence similarity to an earlier Hangzhou sequence.

As of these deposits, the Neuraminidase of emergent ΣH7N9 now has 5 instances of 305V, an amino value found as wildtype in human pH1N1 and in avian H1N1. Furthermore, the amino value is wildtype in H5N1, H6N1 and H9N2, including human cases.

. . . . . . . . 305V [304V],
. . . . . . . . . . . . [Emergent H7N9 ChinaZhejiangHZ1_2013_04,
. . . . . . . . . . . . . . . . . . . . . . . . ChinaHangzhou3_79M_2013_04_02_s [Analysis],
. . . . . . . . . . . . . . . . . . . . . . . . ChinaNanjing1_2013_03_28,
. . . . . . . . . . . . . . . . . . . . . . . . envChinaShanghaiS1088_2013_04_03,
. . . . . . . . . . . . . . . . . . . . . . . . ckChinaShanghaiS1053_2013_04_03],
. . . . . . . . . . . . [pH1N1 WildType
. . . . . . . . . . . . . . . . . . . . . . . . . with codon GTt [107] Re-Emergence North America:
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2012 Toddler & Adult TamiFlu Resistance],
. . . . . . . . . . . . [avH1N1 WildType],
. . . . . . . . . . . . [H5N1 WildType],
. . . . . . . . . . . . [H6N1 WildType],
. . . . . . . . . . . . [H9N2 WildType]

. . . . . . . . 345I [341I],
. . . . . . . . . . . . [Emergent H7N9 ChinaZhejiangHZ1_2013_04,
. . . . . . . . . . . . . . . . . . . . . . . . ChinaHangzhou3_79M_2013_04_02_s [Analysis],
. . . . . . . . . . . . . . . . . . . . . . . . ChinaNanjing1_2013_03_28],
. . . . . . . . . . . . [pH1N1 Australia 2009-11],
. . . . . . . . . . . . [H5N1 LAu China ostrich 2003,
. . . . . . . . . . . . . . . . . . . . China anas 2001]

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & from GISAID?s EpiFlu? Database on which this research is based. An additional list is detailed in the linked PDF entitled "GISAID_Citations_H5N1_2011" at Is H7N9 Spreading from Human to Human in China? Post#164

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case
• Receptor Binding Site Novelty from First Taiwan H7N9 Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

New England Journal of Medicine
Retraction of HA G228S Claim
without
Erratum

GeneWurx comments were originally written on an earlier thread and in earlier posts on this thread concerning a joint production of various CDC units of China that was web published on 2013-05-22 in the New England Journal of Medicine Correspondence entitled Live-Animal Markets and Influenza A (H7N9) Virus Infection:

While we are grateful and relieved to see the Chinese CDC (joint team) remove the claim of HA G228S from the peer-reviewed New England Journal of Medicine discussion, we are also dismayed that no erratum is notated nor any versioning indicated so that those who were mis-led by the error may be suitably relieved of their first impression concerns and may properly cascade their retractions. The small print at NEJM does, however, indicate that an update took place on May 23, but does not document the subject matter or attribution of the revision.

This letter was published on May 22, 2013, and updated on May 23, 2013, at NEJM.org

Narrative

From Page 1, Paragraph 4, Sentence 3 of the original paper (emphasis ours):

Original Publication

Both isolates had potentially functional amino acid sites related to mammal- or human-adapting substitution T189A, Q226L, and G228S (H3 numbering) in the receptor-binding site of hemagglutinin.

Current Web Image

Both isolates had potentially functional amino acid sites related to mammal- or human-adapting substitution T189A and Q226L (H3 numbering) in the receptor-binding site of hemagglutinin.

BP2 versus PB2

If interest remains toward accuracy, the authors may wish to involve their scribes in reading the next sentence after today's correction. Making another update there will add to readability of the segment name that carries the very important mammalian determinant at Polymerase Basic 2 aa627. BP2 should, perhaps be restated as the more common abbreviation, "PB2"? Secondarily, it's never too late for the editorial team at NEJM to have a reading of the full text.

From Page 1, Paragraph 4, Sentence 4 of the updated paper (emphasis ours):

The two isolates showed E rather than K at position 627 of BP2.

PB2 627E has been and remains the standard amino for non-mammal-hosted emergent H7N9, so finding that value in a poultry cage should be expected and is not remarkable. On the other hand, their finding of a human sequence without PB2 627K or PB2 701N was NOVEL and is most certainly a matter of international interest though the specimen was taken from an upper respiratory location. Broncho-alveolar lavage, the gold standard for emergent H7N9, may have produced a more representative Influenza organism with PB2 mammalian adaptations.

No explanation by any of the joint authors from the various Chinese CDC organisations has been added to the discussion concerning the extensive delay in publication of these sequences.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

The two referenced Nanjing sequences have been made available at GenBank now and do NOT show HA G228S.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Two Months
is
Too Long

These GeneWurx comments were originally written on an earlier thread concerning a joint production of various CDC units of China that was web published on 2013-05-22 in the New England Journal of Medicine Correspondence entitled Live-Animal Markets and Influenza A (H7N9) Virus Infection:

Summary

A finding of 228S on the emergent human H7N9 Hemagglutinin, an experimental potentiator of human transmission, apparently de-motivated the authors to publish immediately. Intentionally withholding this data, from a hospitalisation on 2013-03-23 and an admitted tracheal sample taken on 2013-03-28, until now is clearly faulty thinking if, in fact, the reported genetic results are validated. The paper appears to have been written some time ago because the analyses, including the phylogenetic trees, are missing current sequences for comparative purpose.

Aged Phylogenetic Trees

Figure 1. Phylogenetic Tree of Full-Length Matrix Protein (M) Gene of Novel H7N9 Virus. (clickable from original paper) caption (emphasis ours):

The two isolated viral strains of H7N9 — Env/Nanjing and Nanjing/1 — had a remarkable degree of separation from the three other available human strains (Shanghai 1, Shanghai 2, and Anhui 1). Phylogenetic analysis showed that the M genes in the novel virus were derived from avian H9N2 viruses, among others.

The authors did create the illusion of currency by updating the first paragraph with case count and fatality count statistics prior to publication, but the phylogenetic tree caption in Figure 1 and the trees for all segments discussed in the supplement indicate a very early date of production. The comparative set of 2 early Shanghai and the single Anhui human sequences represented the full, public human set for only 5 days, from 2013-03-31 until 2013-04-04. On April 5, 2013, ChinaHangzhou1_C1_38M_2013_03_24_f was deposited at GISAID, a sequence sampled only four days before the Nanjing1 sequence that is now being profiled today for the first time (New England Journal of Medicine).

Furthermore, we would estimate that the authors of this current paper had earlier access than the public to all sequences produced by the CDC units of China. At any rate, the comparatives found on their trees (if restricted to public data) are suggestive of an analytic run prior to April 5, 2013, more than 6 weeks prior to the publication.

Viral researchers and epidemiologists are well aware of what can happen in 30 days during a pandemic emergence. Perhaps we should take caution prior to our next standing ovation for the ostensible transparency and cooperation of these organisations that withhold critical data at inflection points of human history. Who, in the public health leadership, would possibly advise a group of bench scientists to withhold such data? And who in that group of bench scientists would ever allow such information to be withheld?

The narrative indicates HA 228S, but the actual chart of polymorphisms per segment found in the supplement shows that the two sequences under discussion do NOT have the 228S, but remain at 228G wildtype? The Env/Nanjing and Nanjing/1 columns next to HA G228S are notated as 'G', the same as all other H7N9 sequences.

Narrative

From Page 1, Paragraph 4, Sentence 1 of the original paper (emphasis ours):

Two viral strains, one from the patient (Nanjing/1) and one from poultry-cage specimens obtained in the neighboring stall (Env/Nanjing), were successfully isolated (GenBank numbers, KC896771-KC896778 for the case specimen and KC896763-KC896770 for the environmental specimen).

From Page 1, Paragraph 4, Sentence 3 of the original paper (emphasis ours):

Both isolates had potentially functional amino acid sites related to mammal- or human-adapting substitution T189A, Q226L, and G228S (H3 numbering) in the receptor-binding site of hemagglutinin.

Data in Supplement

From Page 12 of the supplement document (labeled 'Page 11'), we have excerpted the lines of interest (emphasis ours, dots employed for web spacing):

Table S1. Analysis of the Key Amino Acid Positions in Different Viral Proteins Related with Interspecies Transmission or Drug Resistance.

. . . . . . . . . . . . . . . . . . . . . . . . . . . Env/Nanjing Nanjing/1 Shanghai/1 Shanghai/2 Anhui/1
HA Receptor binding site (RBS) G228S . . . . . G . . . . . . . G . . . . . . G . . . . . . G . . . . . G . . .

Is this disjunction between the narrative and the actual data yet another publication error in a peer-reviewed journal concerning this potential H7N9 pandemic emergence? Does the peer-review process, at some point, involve a reading of the paper?

The sequences are not yet available at GenBank for verification.

We acknowledge the authors, originating and submitting laboratories of the sequences from GenBank & from GISAID’s EpiFlu™ Database on which this research is based. An additional list is detailed in the linked PDF entitled "GISAID_Citations_H5N1_2011" at Is H7N9 Spreading from Human to Human in China? Post#164

GISAID Citations

• GeneWurx Cross Serotype Homology Analysis, Open-Access, Full-Text version
• Human Emergent H7N9 from Zhejiang Province
• Environment Emergent H7N9 from Zhejiang
• Fatal H5N1 Homology to Emergent H7N9 from Shanghai in March
• Fatal H5N1 Karo Cluster Homology to Emergent H7N9 from First Fujian Case
• Receptor Binding Site Novelty from First Taiwan H7N9 Case
• China - H7N9 Human Isolates on Deposit at GISAID, Comprehensive Genetics Discussion