China - H7N9 Human Isolates on Deposit at GISAID

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

"RoundTrip" FlightPath

A grateful acknowledgement to the researchers and GISAID team who have resolved the data transmission issue and removed the duplicate H7N9 sequence as of 2013-04-11-20:00, instantly making this request obsolete.

By appearances, even the human sequences can fly.

A Duplicate "RoundTrip" H7N9 isolate of ChinaHangzhou1_C1_38M_2013_03_24_f was posted at GISAID today from GenBank. H7N9 Count at GISAID is now at 35, although duplicates reduce the actual distinct set sequences and the accuracy of the database. De-Duplication and "house-holding/connecting" are mainstay early design imperatives among Big Data projects involving data-sharing between disparate organisations.

Statistical anomalies prevail in a database without curation.

This apparent 20% increase in sample size (Human cases from 4 to 5) is a substantial variation for most calculations. And isn't that where we're all focused . . . on the variation? But we need to see ACTUAL variation, not skewing due to avoidable data quality issues.

Because we've seen the resounding negative effects of data duplication in critical scenarios, a request for information concerning the maintenance schedule for this ongoing issue has been made to GISAID.

At this stage of the H7N9 species transition where acceleration is probable, taking this opportunity now to correct these data transmission errors may indeed provide the footing to tame the transmission of the actual disease. Some will say that data is always dirty, perpetually imperfect. But we all know that with a little work, like this devoted FT forum, most data can be improved and some data even shows the upside of being perfected.

And I think we all can agree that the Power of Perfect Information is ineffable.

[camster]

Re: China - H7N9 Human Isolates on Deposit at GISAID

I would dearly like to see sentinel flu lab data for "Type A - unsubtyped" returns. When samples are RT-PCR tested it is against supplied primers one of which tests for a match against conserved areas on the 'M' RNA strand which is common to all type A flus. If it passes this test then it is checked for a match against seasonal flu markers, which it would fail. If there have been other human H7N9 infection in the general ILI background then they should show up as a spike in the "Type A - unsubtyped" returns. This is important data we are missing and, if the samples have been retained, a prime candidate for retesting once the H7N9 primers have been sent out to the labs in the sentinel system (of which I believe there are about 600).

This is my 2 cents, but I think this is one of those rabbit holes you can dig yourself into. The fact of the matter is that the universal influenza A tests have a higher (often up to 10-100 fold) sensitivity than the sub-typing tests, so if you're near the limit of detection (which you often are for a number of reasons: poor swabs, lousy patients etc.), you'll get a Flu A positive/unsubtyped result. What you're really looking for are samples with low CT scores on the flu A test (=high titer) that don't subtype, and those are exceedingly rare and supposed to be chased down.

One other thing I see often is the suggestion that because an avian strain isolated from humans has some 'human' markers, that the new virus must have had some sort of contact with a human strain to gain that mutation. These viruses have sloppy polymerases that generate a lot of diversity on purpose. The reported sequences generated from all these samples represent the majority vote at each position, but it doesn't tell the whole story. The whole-genome guys have started to represent this data as 'probability fields'. That is, you might have an A at a position 98% of the time, but the other 2% of time it could be C, G or T. BUT it's there at some low level. We did some admittedly sloppy calculations over lunch one day and came to the conclusion that any human with a reasonable influenza infection not only has every single possible amino acid substitution present in their nasal cavity, but quite likely every single DOUBLE MUTATION as well.

What that means is that the individual virus that was lucky enough to have the mutation(s) that allow it to function well in a new host is the one that successfully transfers and gets amplified. At that point, the mutation is fixed in the new population. Its your most basic evolutionary selection event and the reason those sloppy polymerases are kept, it helps the virus find new hosts. No cross-training required!

cheers,
camster

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

This is my 2 cents, but I think this is one of those rabbit holes you can dig yourself into. The fact of the matter is that the universal influenza A tests have a higher (often up to 10-100 fold) sensitivity than the sub-typing tests, so if you're near the limit of detection (which you often are for a number of reasons: poor swabs, lousy patients etc.), you'll get a Flu A positive/unsubtyped result. What you're really looking for are samples with low CT scores on the flu A test (=high titer) that don't subtype, and those are exceedingly rare and supposed to be chased down.

One other thing I see often is the suggestion that because an avian strain isolated from humans has some 'human' markers, that the new virus must have had some sort of contact with a human strain to gain that mutation. These viruses have sloppy polymerases that generate a lot of diversity on purpose. The reported sequences generated from all these samples represent the majority vote at each position, but it doesn't tell the whole story. The whole-genome guys have started to represent this data as 'probability fields'. That is, you might have an A at a position 98% of the time, but the other 2% of time it could be C, G or T. BUT it's there at some low level. We did some admittedly sloppy calculations over lunch one day and came to the conclusion that any human with a reasonable influenza infection not only has every single possible amino acid substitution present in their nasal cavity, but quite likely every single DOUBLE MUTATION as well.

What that means is that the individual virus that was lucky enough to have the mutation(s) that allow it to function well in a new host is the one that successfully transfers and gets amplified. At that point, the mutation is fixed in the new population. Its your most basic evolutionary selection event and the reason those sloppy polymerases are kept, it helps the virus find new hosts. No cross-training required!

cheers,
camster

Thank you for introducing the quasi-species / probability fields concept that is a most certain fact. Understanding that the sequencing output is more an approximation than a comprehensive evaluation is essential to governing our expectations of the data.

[Catbird]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Belated but sincere thanks JJackson. Your explanation helped to clarify some things for me.

[JJackson]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Camster thank you so much for taking the time to add some depth to my rather superficial understanding of the realities of testing. In light of your post then what I really want to see is if their has been an uptick in the samples with a low CT score that do not subtype, which would weed out any weak samples that were just strong enough to score a hit on the type A test but too weak to give a positive against a serotype specific primer.

Regarding the second paragraph I find it helpful when trying to understand what is going on in a host to think of it infecting individual cells. Transmission to a new host is very much the exception to the rule. For every released virion 99.99% are going to infect another cell in the same host the one(s) that seed a new host are very much a Columbus or Vasco da Gama. As many generations of infection, replication and reinfection are taking place in the same host there is also plenty of scope for the generation of new reassortments, recombinations and SNPs. Unless a particular change keeps cropping up in different hosts I normally work on the assumption it has been generated locally and is just a normal by-product of the quasi-species aspect of flu. As flu, like all viruses, is an obligate parasite the cost of producing all those dodgy copies is born by the host so it is no great loss to the virus if its RNP is somewhat lacking in quality control.

[gsgs]

Re: China - H7N9 Human Isolates on Deposit at GISAID

what's the diversity in segment 5, when did it reassort

[AlaskaDenise]

Re: China - H7N9 Human Isolates on Deposit at GISAID

..........What that means is that the individual virus that was lucky enough to have the mutation(s) that allow it to function well in a new host is the one that successfully transfers and gets amplified. At that point, the mutation is fixed in the new population. Its your most basic evolutionary selection event and the reason those sloppy polymerases are kept, it helps the virus find new hosts. No cross-training required!

cheers,
camster

Thanks for the explanations. Tis good to read the implications of a promiscuous pathogen.

Might the "fixed mutations" vary within an individual host, depending on the immune genetics of any one part of the body?, e.g., the P53 gene can be active in one area and silenced in another, thereby influencing the virus' ability to replicate differently in different locations, as the NS1 segment interacts with downstream parts of P53 which control growth arrest and apoptosis.

.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Are any of the cases gravid?

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Potential H5N1 Involvement
in H7N9 Hemagglutinin Emergence

This 541 residue is highly conserved in H7 as Alanine and is within a highly conserved region. The Valine revision located on all emergent H7N9, except the first human fatality, is also recently seen as a polymorphism on Human H3N2, South Africa 2012-06. More importantly, the value appears to be Novel to H7, but is found widely in circulating avian H5 (H5N1, H5N2, H5N3, H5N7, H5N9) and many human H5N1 cases from Asia (Cambodia, Hong Kong, Thailand and Vietnam). Tigers and leopards have also carried this value in H5N1 alongside more than 50 species of birds.

. . . . . . . . 541V [533V H5N1 wildtype],
. . . . . . . . . . . . [sH3N2 South Africa 2012]

• Ongoing Sequence Analysis - Open-Access, Full-Text
• GISAID Citation

[AlaskaDenise]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Just checked GISAID and found that the nice folks at Harbin Vet. Research Institute deposited a sequence from a Shanghai chicken.

The NS1 103 & 106 are showing what Fouchier wrote increases air transmission in ferrets.

Another paper, The Virulence of 1997 H5N1 Influenza Viruses in the Mouse Model Is Increased by Correcting a Defect in Their NS1 Proteins, explains that while both the non-concensus values of L&I and the concensus values of F&M both are highly pathogenic, one causes more rapid onset with spread to other organs (like the brain), while the other causes more severe lung damage. I have had to reread this paper several times, so please read it for yourself as I don't think I could summarize it well.

This may be an important characteristic of H7N9/2013.

.

[AlaskaDenise]

Re: China - H7N9 Human Isolates on Deposit at GISAID

AND.... Harbin (Huihui Kong) shared a.... pigeon on GISAID!
Perhaps next they'll share one of those quails.

Now we can all await more analysis by the global scientific community.

The NS1/103 & 106 has the same values as the chicken and humans.

The pigeon HA is most similar to people and chicken in China, but then a Korean wild bird, Mongolian duck, and poultry from Europe???

Hopefully this newest data will help more scientists put their collective minds to work on this newcomer virus.

.

[AlaskaDenise]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Using one Shanghai human H7N9, I did a blast on each segment to look for similarites for mammals (mostly humans). The NA was mostly like wild birds and the other segments were mostly like domesticated poultry, with a few swine, equine, and canine similaries. There were also some quail (with receptors for birds and humans), however there were more quail similarities in the MP, M1, & M2. In the NP there were similarities to 2 human cases of H5N1 in 2007. This paper discusses the importance of some locations in the NP to establish dominance of a sublinage, in H5N1, but is also in human H1N1 - both seasonal and 2009 pandemic. The new 2013 H7N9 has those same NP dominance values.

.

[AlaskaDenise]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Yoshihiro Kawaoka1,3,5,6,7

- Author Affiliations

1Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan


3Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin?Madison, Madison, Wisconsin, USA


5International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan


6Division of Zoonosis, Department of Microbiology and Infectious Diseases, Graduate School of Medicine, Kobe University, Hyogo, Japan


7ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan

http://vir.sgmjournals.org/content/92/7/1645.full

.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

AND.... Harbin (Huihui Kong) shared a.... pigeon on GISAID!
Perhaps next they'll share one of those quails.

Now we can all await more analysis by the global scientific community.

The NS1/103 & 106 has the same values as the chicken and humans.

The pigeon HA is most similar to people and chicken in China, but then a Korean wild bird, Mongolian duck, and poultry from Europe???

Hopefully this newest data will help more scientists put their collective minds to work on this newcomer virus.

.

Blazing the Origin Trail

As of 2013-04-01, the quail became a very important species of interest for GeneWurx, practically a keystone.

Although we cannot yet quantify the biochemical mechanism, the current working hypothesis is that quail, either biologically or via culture, is distinct, perhaps in a vein similar to the gulls with H13 (without the known statistical intensity). We would appreciate thoughtful deliberation from ornithologists, wildlife management specialists, domestic poultry specialists including avian veterinarians, avian viral receptor experts and others who may have connective information:

• What makes a quail different?
• What are the quail cell receptor type ratios by comprehensive tropism?
• What are the quail habitat overlaps / interactions with goose and duck species in the wild and in captivity?
• Is the chukkar / partridge of a similar characteristic set?

Focus on the quail species with subsequent study inclusion occurred when polymorphic signals demonstrated a skewed homology rate between emergent H7N9 and an avian segment from the previous decade, Ancestral Quail (aQγ). Though the serotypes differ, a statistically significant dispersion (~40 aminos) of the H7N9 HA polymorphisms is found on the aQγ segment. That aQγ HA appears to have accepted polymorphisms at the sub-segment level of known H7N7 mammal adaptations and to have had extensive interchange with serotypes from both classes of Influenza A.

HA Shared Homology (aQγ + Emergent H7N9)

• Human and avian H5N1
• Human and avian H3N2 High-VxX
• Human pH1N1.Upsilon High-CFR

The gain of mammalian genetic traits make the aQγ segment distinct from the geographically and temporally-correlated avian series (n=6 inclusive).

HA Genetic Distinction (aQγ)

• ~059 base Δ to Nearest specimen within related Avian Series
• ~075 base Δ to Most distant specimen within related Avian Series
• ~187 base Δ to Nearest specimen BEYOND related Avian Series (6th Closest Relative)

Suggestion of co-infection is exhibited in the specimen. The greater portion of the aQγ overlapping homology to emergent H7N9 occurs at the 3' end of the HA.

A rather severe gap in the data record exists after that distinct aQγ arrival. Limited signals occur earlier and later in the record, but population count is far too low to discern decision-making information / heuristic determinants. Having identified this earliest known emergent H7N9 ancestral donor, we would expect to find several dozen step-wise, intermediate Hemagglutinin segments were the Influenza database more robust. While we do not consider this quail to be the Ultimate Origin of emergent H7N9 or even penultimate for that matter, the homology level gauges a solid tertiary or gamma plateau ranking until further data and / or analysis proves otherwise.

A functional data store, perhaps at GISAID, built from sequencing the avian H7N9 samples languishing in cryo would be a very well-received investment by the world's wildlife and public health conservatories.

Activating budget alternatives now is certainly supported by the epidemiology, the genetic trend and the risk projection scenarios.

[NS1]

Re: China - H7N9 Human Isolates on Deposit at GISAID

Using one Shanghai human H7N9, I did a blast on each segment to look for similarites for mammals (mostly humans). The NA was mostly like wild birds and the other segments were mostly like domesticated poultry, with a few swine, equine, and canine similaries. There were also some quail (with receptors for birds and humans), however there were more quail similarities in the MP, M1, & M2. In the NP there were similarities to 2 human cases of H5N1 in 2007. This paper discusses the importance of some locations in the NP to establish dominance of a sublinage, in H5N1, but is also in human H1N1 - both seasonal and 2009 pandemic. The new 2013 H7N9 has those same NP dominance values.

.

Great work, Denise!

The internal segments are lightly evaluated for mammal and zoonotic involvement on post #10.