Debate rages over new bird flu research; some argue it’s not safe to publish

[Kassy]

http://www.ctv.ca/CTVNews/Health/201...tudies-120218/

The research will be published after some more delays.

[Kassy]

Commentary

Detail On Fouchier H5N1 Transmission Paper
Recombinomics Commentary 17:30
February 22, 2012


Ron Fouchier and colleagues at the Erasmus Medical Centre in Rotterdam, the Netherlands, created transmissible H5N1 by putting two mutations into the HA surface protein of an H5N1 virus, and one into its polymerase enzyme, before exposing ferrets to it. It spontaneously acquired two more significant mutations, and remained lethal.

The above comments strongly suggest that the H5N1 that transmitted in ferrets began with two receptor binding domain changes, Q226L and G228S, as well as PB2 E627K. Passage in ferrets selected two more changes with S227N and Q196R at the top of the list of likely acquisitions, based on a CDC publication in Virology, "In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity"..

The CDC paper looked at quasi-species in A/Vietnam/1203/2004, which had a number of receptor binding domain changes, including S227N and Q196R which synergized with each other. However, Q196R, when combined with two receptor binding domain changes seen in prior pandemics, Q225L and G228S, produced a dramatic change in receptor binding, favoring 2,6 receptors which are present at high levels in human (and other mammal) upper respiratory tracts. This combination led to detectable H5N1 in the upper respiratory tract of ferrets. However, to achieve efficient transmission, the above construct was placed on a clade 2.2 H5 background, and 6 H5N1 genes from A/Vietnam/1203/2004, which contained PB2 E627K (which was also in the clade 2.2 isolate from Egypt, and the N1 was replaced with an N2 from seasonal H3N2 (A/Brisbane/10/2007). This construct transmitted in ferrets.

Thus, the Fouchier result demonstrated that the requirement for a clade 2.2 genetic background, as well as a seasonal N2 could be eliminated by two changes on H5 when clade 2.1 from Indonesia was used, indicating the creation of a transmitting H5N1 was easier than changes noted in the CDC paper.

Similarly, the Kawaoka paper showed that H1N1pdm09 genes could substitute for the human N2 and the PB1 E627K.

The results confirm that H5N1 transmission requires a minimal number of changes, and reproduction of the results described in the Science and Nature papers can be easily achieved with the public information described above.

http://www.recombinomics.com/News/02...er_Detail.html

[Sonny]

Originally Posted by Kassy

Commentary

Detail On Fouchier H5N1 Transmission Paper
Recombinomics Commentary 17:30
February 22, 2012

Kassy, who's avatar looks like an Ebola virus.
Does this novel Ha5 have compelling potential as a pre-pandemic strain?
And If so is this one different enough that we'll need a new vaccine,if this is the one?

[Kassy]

Probably since the mutations the Fouchier group used are ones occuring normally. I wish they would publish the thing because then we can look at the details and check how often we find combinations of the five mutations in the historical Sequences.

Above it is said the first 3 mutations induced to more so it would be most interesting to see how many of the first group were found together. Do we have sequences with all three? How many with two?


There are vaccines for H5N1 around. They can easily test if they work for the Fouchier strain with another bunch of ferrets.

They might be doing that now. If too many ferrets die we need a new vaccine but maybe the old one works.

[Kassy]

Similar research on H5N1 has been published recently without biosecurity ruckus.
I guess the simple elegance of the Fouchier research triggered that:

Diverse Ferret H5N1 Transmission Pathways

Recombinomics Commentary 21:00
February 22, 2012


However, a paper that shows how to makes H5 readily transmissible had already been published in September last year by Reuben Donis and colleagues at the US Centers for Disease Control and Prevention in Atlanta, Georgia. His team equipped a different H5N1 with the same two mutations as Fouchier. He also added a third mutation, and switched other genes to make it transmissible among ferrets.

The above comments add considerable clarity for the requirements for efficient transmission of H5N1 in a ferret model. The above CDC paper has been published and therefore the receptor binding domain changes and associated influenza backgrounds are known. The transmitting H5N1 had 3 receptor binding domain changes (Q196R, Q226L, G228S), and two have been discussed in tandem for years, so it is virtually certain that the two RBD changes introduced by the Fouchier lab were Q226L and G228S. Similarly, the two viruses used in the above study, A/egret/Egypt/1162/2006 and A/Vietnam/1203/2004 have PB2 E627K, which is known to increase virulence and transmission in mammals, so the three changes introduced by the Fouchier lab are almost certainly Q226L and G228S in HA and E627K in PB2.

Thus, these two studies used very different approaches, but overlapped in at least three of the changes in the generation of a transmitting virus. The Fouchier study indicated the creation of a transmitting H5N1 was far less complex than the CDC study, which used a clade 2.2 H5, a seasonal H3N2 (A/Brisbane/10/2007) N2, and clade 1 internal genes. Fouchier used one H5N1 from Indonesia (clade 2.1) and only added three well known changes. Moreover, it is likely that the two additional changes were also well known, and may be among the other changes studied by the CDC including Q196R and S227N which synergize with each other, while Q196R in combination with Q226L and G228S drives the receptor binding to gal 2.6.

Similarly, the Kawaoka lab used a third approach, which took the H5 gene and put it on an H1N1pdm09 genetic background. The H5 was from Vietnam and was almost certainly another sub-clade (2.3.4.2), which demonstrated the versatility of the H5N1 H5, as well as the other genes which could support transmission. Thus, in addition to the H5, one lab used a different clade for internal genes (which also supplied E627K) and a human N2, while the other lab used H1N1pdm09 for the internal genes as well as N1.

It is unclear if additional receptor binding domain changes were introduced by the Kawoaka, but it is clear that three different H5 sub-clades can transmit when supported by diverse additional flu genes, highlighting the need for additional study in this area.

Moreover, it si also clear that the delay in publication of these studies servers no purpose, since the three changes introduced by Fouchier are clear, and passage of influenza in ferrets has been done for decades.

H5N1 remains a poor choice as a bioweapon, because it can’t be controlled once released, and the three experiments described above indicate efficient H5N1 transmission can be generated using a very wide array of combinations, highlighting the need for an aggressive H5 vaccination effort.

http://www.recombinomics.com/News/02...Diversity.html

So we have 3 examples where the same changes in Ha and PB2 are enough to support a different set of other "internal" genes.

[Kassy]

Commentary

Similarities In CDC and Fouchier H5N1 Transmission Papers

Recombinomics Commentary 12:00
February 23, 2012


A reverse genetics virus with HA of EG06-196R/226L/228S, the N2 neuraminidase from a human seasonal H3N2 virus, A/Brisbane/10/2007, and 6 internal genes from a clade 1 H5N1 virus, VN04, known to replicate efficiently in ferrets (Maines et al., 2011), was generated and evaluated in the ferret transmission model. As shown in Table 2, both direct contact ferrets that were housed in the same cage with inoculated ferrets became infected, as evidenced by virus shedding and seroconversion results. In addition, viral shedding was also detected in one of two ferrets housed in adjacent cages and exposed only via respiratory droplets.

The above comments are from the CDC paper, “In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity", published online on November 5, 2011 in the journal Virology. As noted above, the three receptor binding domain changes included the two most noted changes in H5N1 transmission discussions, Q226L and G228S, along with Q196R which was present in H5N1 clusters in Iraq and described in previous studies by Kawaoa. These changes were placed on a clade 2.2 H5 (A/egret/Egypt/1162/2006) which was pl aced on a genetic background using human N2 (A/Brisbane/10/2007) 6 H5N1 clade 1 genes (A/Vietnam/1203/2004), which includes PB2 E627K. This combination led to H5N1 transmission in ferrets via respiratory droplets as stated above.

Thus, the “recipe” for H5N1 was published in full in a scientific journal which could be accessed by anyone with an internet connection. It provided full detail on the changes, which have been described in detail previously. Moreover, it used well known isolates which had been sequenced and were publicly available at Genbank or GISAID.

Thus, after the details were published describing the US CDC H5N1 transmission results, the US NSABB requested papers submitted to Nature and Science be delayed and censored. Ironically, the CDC paper was more of a recipe for a bioterrorist because it cobbled together a virus that was unlikely to be created in nature because clade 2.2 is now largely limited to Egypt, clade 1 has never been reported outside of southeast Asia, and the assembled virus included a human N2 gene so technically the transmitting H5N1 was really H5N2.

In contrast, the Fouchier paper, which is delayed at Science, started with changes present in the CDC published paper. It had the two most publicized RBD changes, Q226L and G228S, in HA, as well as the most publicized polymerase change, PB2 E627K. The Fouchier paper placed these three well known changes on an Indonesia clade 2.1 genetic background, which also achieved ferret transmission, while maintaining lethality. Passage in ferrets led to the selection of two additional well known changes, and these two changes are of interest to the scientific community, but of little interest to a bioterrorists, since passage in ferrets is a well known technique which would select the two additional changes.

The Kawaoka paper also used a combination that was more likely to be generated via natural selection because he used a clade 2.3.4.2 H5 from Hanoi (almost certainly A/Vietnam/HN31604/2009) which was placed on an H1N1pdm09 genetic background (A/California/04/2009). It is unclear if he also created Q227L and G228L, but it is well known that H1N1pdm09 PB2 can substitute for H5N1 PB2 with E627K to produce efficient human transmission, and H1N1pdm09 is widespread in humans, swine, and turkeys which provide natural opportunities for the creation of an H5 reassortant with a composition that matches the H5N1 used by Kawaoka in his paper in Nature.

Thus, the two delayed papers demonstrate that a subset of the changes used in the published CDC paper, which were on a clade 2.2 H5 from Egypt, could be placed on a clade 2.1 H5 from Indonesia to create droplet transmission in ferrets, which can also be achieved using a clade 2.3.4.2 H5 from Vietnam (on an H1N1pdm09 background).

Therefore, the NSABB request to redact detail in the Fouchier and Kawaoka papers had no scientific basis, and was appropriately rejected by the scientific community familiar with the detail. The papers at Nature and Science should be published immediately, and the NSABB should get a new board that is more knowledgeable about H5N1 evolution and reasons why a transmitting H5N1 would not be a good choice as a weapon of mass destruction.

http://www.recombinomics.com/News/02...ssion_CDC.html

[Kassy]

http://www.nature.com/news/the-risks...tudies-1.10138

Nature articles with details.

http://www.newscientist.com/blogs/sh...ef=online-news

And New Scientists.

[Kassy]

Older article where Fouchier and others look at the minimal requirements for airbourne transmission in influenza:

http://www.sciencedirect.com/science...7962571100054X

[Kassy]

Commentary

Full Fouchier Formula for H5N1 FerretTransmission
Recombinomics Commentary 09:00
March 3, 2012

Actually, though, it's the wild-type virus that's odd. In general, when you give ferrets most kinds of H5N1 in their noses, said Fouchier, "they don't get sick at all". The strain he used, collected in 2005 in Indonesia, is known for causing unusual brain-related disease when put in ferrets' noses. Perhaps the mutant has lost this: the ferrets who got it in their noses, either by breathing it in or because it was put there, just got flu.
The above comments, as well as additional remarks, indicate the H5N1 used in the Ron Fouchier ferret transmission studies was A/Indonesia/5/2005 from the first confirmed human cases in Indonesia and the WHO clade 2.1 vaccine target. The use of this H5N1 in prior ferret studies was described in a Fouchier 2011 American Journal of Pathology paper, “Pathogenesis of Influenza A/H5N1 Virus Infection in Ferrets Differs between Intranasal and Intratracheal Routes of Inoculation”. That paper described different pathologies linked to various inoculation routes of A/Indonesia/05/2005, which match the descriptions in the recent report.

Thus, the Fouchier clade 2.1 Indonesian H5N1 virus could kill ferrets and cause neurotropic effects, but the transmitting H5N1 did not kill ferrets. These results are similar to another published paper, which used a 2006 clade 2.2 virus from Egypt. That CDC paper gave detailed descriptions for the creation of an H5N1 that also transmits in ferrets. Similarly, the Yoshi Kawaoka paper delayed at Nature uses an H5 from another H5N1 sub-clade (2.3) which was placed on an H1N1pdm09 genetic background (as described in a prior publication) to also transmit in ferrets.

Thus, the full recipe for two of the three transmission studies is now known, as is the general formula for the third study. Fouchier took a 2005 clade 2.1 isolate, A/Indonesia/05/2005, added three (HA Q226L, G228S; PB2 E627K) of the four changes in the CDC paper, and passaged the H5N1 in ferrets 10 times. The CDC took a 2006 clade 2.2 isolate, A/egret/Egypt/1162/2006, added three HA changes, Q196R, Q226L, G228S and placed it on a clade 1 genetic background, A/Vietnam/1203/2004, which had PB2 E627K, and added seasonal N2 gene from A/Brisbane/10/2004. Kawaoka took a 2009 clade 2.3 isolate, A/Vietnam/HN31604/2009, placed it on an H1N1pdm09 background (A/California/04/2009) and probably added the same two HA changes used in the Fouchier and CDC papers (Q226L and G228S).

Recent comments by some of the NSABB board members clearly demonstrates a profound lack of understanding of the published data, which is why the papers should be published in full immediately, and the NSABB board should be replaced with scientists more familiar with the scientific literature on H5N1 transmission.


http://www.recombinomics.com/News/03...hier_Full.html

[Kassy]

Both papers are now officially OK'd by the NSABB.

News:
http://www.nydailynews.com/life-styl...icle-1.1054495

http://blogs.discovermagazine.com/no...26+Medicine%29

And and upcoming symposium is goint to be available as webcasts & there is a link for webcats from a february symposium:

http://www.eurekalert.org/pub_releas...-hrs040212.php

[Kassy]

In all the ruckuss dutch government decided that research involving pathogens needs an export permit and then promptly denied the first one because there was a controversy.

This still hinders publication of Fouchiers' piece even though it seems it will be published now. There's a meeting about the issue but it will be published anyway regardless of the outcome.

http://www.nature.com/news/mutant-fl...ission-1.10469

[Kassy]

http://news.sciencemag.org/sciencein...ion.html?rss=1

Fouchier applied for the export permit under protest & it has been granted now so the article can finally be published.

[Kassy]

http://www.newscientist.com/article/...s&nsref=health

It will be published this week.

New research is not being done awaiting clarifications on new rules & regukations.