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AVIAN INFLUENZA, HUMAN (40): PUBLICATION EMBARGO LIFTED
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[1]

Date: Fri 30 Mar 2012
Source: New York Times [edited]
http://www.nytimes.com/2012/03/31/healt ... s.html?hpw


After months of debate and public furor over experiments that made a dangerous flu virus more contagious [see ProMED-mail archived reports below], a panel of scientific advisers reversed itself on Friday [30 Mar 2012] and recommended that details of the research be published in full in scientific journals.

The panel, the National Science Advisory Board for Biosecurity, had originally recommended in December [2011] that scientific reports about the research be published only if certain details were withheld, to prevent others from replicating the experiments. The fear was that bioterrorists would use the information to create germ weapons. There was also concern by some scientists that the virus would leak out of a laboratory by accident and start a pandemic.

The December [2011] recommendation was the 1st time that the biosecurity panel had ever advised keeping details of biological research secret. In a statement issued on Friday [30 Mar 2012], the board said that the reports had been revised and that the data in them "did not appear to provide information that would immediately enable misuse of the research in ways that would endanger public health or national security." In addition, it said new data has emerged indicating that understanding the precise genetic changes that increase contagiousness in flu viruses could improve scientists' ability to monitor viruses in the environment and identify those with the most potential danger.

The experiments involve a type of bird flu virus known as H5N1 [avian influenza A/(H5N1) virus]. It does not often infect people but appears unusually deadly when it does. People almost never transmit the virus to one another, but if the virus were to become transmissible, deadly pandemics could ensue. The experiments in question studied the virus in ferrets, considered a good model for the way flu viruses affect people. In the course of studying transmissibility, the research teams created a strain of the virus that could spread through the air from one animal to another, something that had not existed before.

The work, paid for by the National Institutes of Health, was done by 2 separate research teams, at Erasmus Medical Center in Rotterdam, in the Netherlands, and at the University of Wisconsin-Madison. In January [2012], the scientists voluntarily suspended research on the viruses because of the uproar it provoked. The moratorium was to last 2 months, but the World Health Organization said in February 2012 that it would probably be extended for a few more months.

[Byline: Denise Grady]

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[2]
Date: Fri 30 Mar 2012
Source: CIDRAP News [abbreviated & edited]
http://www.cidrap.umn.edu/cidrap/conten ... nsabb.html


A federal advisory board today [30 Mar 2012] reversed its stance on publishing 2 controversial H5N1 transmission papers, recommending that both studies be published in full. The National Science Advisory Board for Biosecurity (NSABB) met yesterday and today [29-30 Mar 2012] to discuss revised versions of the studies by 2 groups, one from the University of Wisconsin, Madison and one from Erasmus University in the Netherlands. Both studies were funded by the National Institutes of Health (NIH), and, as its dual-use advisory board, the NSABB in December [2011] recommended redacting the studies due to bio-terror concerns and the risk of an unintentional release of the viruses used in the 2 ferret studies. The 2 journals slated to publish the studies, Science and Nature, have held off on running the papers based on the NSABB's nonbinding recommendation, which the NIH agreed with in December 2011.

In a statement released today [30 Mar 2012], the NSABB said it unanimously recommended that the revised manuscript by the University of Wisconsin group, headed by Yoshihiro Kawaoka, DVM, PhD, be published in full, and members voted 12 to 6 that the data, methods, and conclusions in the revised paper by the Erasmus group, headed by Ron Fouchier, PhD, be published.

Today's [30 Mar 2012] action by the NSABB follows yesterday's launch of a new government policy on the oversight of federally funded life sciences dual-use research. The new policy requires federal agencies to routinely review the risks of research conducted on 15 "high consequence" pathogens and toxins, including highly pathogenic H5N1 avian flu. In its statement, the NSABB said it strongly supports free communication of research information unless it poses significant and immediate risks to public health and safety. "While the communication of information in these manuscripts still presents dual-use concerns, the additional information changed the board's risk-benefit calculation," it said.

More specifically, the board said that data in the revised manuscripts don't appear to provide information that would immediately enable misuse in ways that would threaten public health. The NSABB also said new evidence has emerged that "underscores the fact that understanding specific mutations may improve international surveillance and public health and safety." The group did not elaborate on the new evidence.

"Global cooperation, critical for pandemic influenza preparedness efforts, is predicated upon the free sharing of information and was a fundamental principle in evaluating these manuscripts," the NSABB added. It concluded, "The NSABB recommendations from this meeting will be forwarded to the US government for review and consideration." The board noted that its recommendations were informed by the new dual-use oversight policy that was announced yesterday [29 Mar 2012]. The group emphasized that better processes for responsibly communicating dual-use research urgently need to be developed. On 3-4 Apr 2012, a Royal Society conference in London will address various aspects of the H5N1 research issues, and the World Health Organization (WHO) has said it will host a 2nd meeting on the H5N1 issues in the middle of the summer [2012].

Kawaoka's team combined the hemagglutinin from an H5N1 virus with other genes from an H1N1 virus and found that the hybrid could spread in ferrets via respiratory droplets, although it did not kill any of them. Fouchier's group used genetic engineering and passaging through ferrets to generate an H5N1 virus that spread via the airborne route among ferrets. Accounts of the virus's lethality have differed somewhat, but it was at least lethal to ferrets with deliberate tracheal inoculation at high doses.

[Byline: Lisa Schnirring]

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[These reports are posted to record final resolution of the long-running controversy around the desirability, or not, of publication of controversial experimental data concerning genetic manipulation of the transmissibility of avian A(H5N1) influenza virus. - Mod.CP]

AVIAN INFLUENZA, HUMAN (42): MUTATIONS AFFECTING TRANSMISSIBILITY
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Date: Tue 3 Apr 2012
Source: CIDRAP (Center for Infectious Disease Research & Policy) News [summ., edited]
http://www.cidrap.umn.edu/cidrap/conten ... royal.html


H5N1 mutations unveiled at H5N1 meeting
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New details about the H5N1 transmission studies emerged today during a Royal Society conference on the topic in London, but with Dutch export restrictions blocking what one research group could share. Scientists who led the 2 studies in question both took the stage during the 1st day of a 2-day meeting to discuss a host of issues on the topic, from basic research to ethics. The Royal Society is offering a live webcast of the meeting. Fresh information about the 2 controversial studies comes just days after the National Science Advisory Board for Biosecurity (NSABB) reversed an earlier recommendation that the studies be published in redacted form, due to concerns about an accidental or intentional release of the modified H5N1 viruses.

Yoshihiro Kawaoka, DVM, PhD, who leads a group from the University of Wisconsin, Madison, gave a detailed look at his team's H5N1 studies on ferrets. He highlighted 4 mutations that his group produced, one of which is found in nature. 3 of the mutations were on the head of the virus HA [haemagglutinin protein] and one was on the stalk. The findings will appear in a future issue of Nature.

He said using a hybrid between H5N1 and the 2009 H1N1 virus allowed his group to focus on the haemagglutinin portion. Kawaoka added that the mutant strains weren't lethal to the animals and that the pathogenicity was less than that of the 2009 pandemic virus. Viruses containing the mutations were highly sensitive to antiviral drugs, and they reacted to sera from humans vaccinated against H5N1 [perhaps this should read sera from infected patients?]. Kawaoka added that his group's data could have implications for public health polity makers. "Our information allows us to find the right virus for the production of stockpiled vaccines," he added. of the mutations -- the one found in nature -- had loss of a glycosylation site, a characteristic that Kawaoka said is linked to transmissibility in humans, and a pattern increasingly seen in countries such as Egypt in which the H5N1 virus has infected humans. "We can use the information to understand what's happening in nature," he said, adding that better surveillance is needed to assess the risk of circulating strains.

Though the other research group, led by Ron Fouchier, PhD, at Erasmus University in the Netherlands, used a different virus strain in their ferret studies, "we found striking similarity -- mechanistically, a very interesting finding," Kawaoka said. However, Fouchier said today [3 Apr 2012] that he could only share the redacted version of his study at the conference, because the Dutch government hasn't yet lifted export controls on the information. While he couldn't disclose the mutations, he said the 2 studies provide more unanimous expert advice to countries that battle H5N1 threats. The group's findings will be published in an upcoming issue of Science.

Though the NSABB's reversal helps clear the way for publication in the 2 journals, the status of a voluntary moratorium on H5N1 research is unclear. In February 2012 a World Health Organization (WHO) expert panel recommended a host of measures, which included a research moratorium, pending a biosafety review of the practices at the labs.

One theme that wove through several of the Royal Society presentations today [3 Apr 2012] is the need for more discussion to tackle issues related to H5N1 transmission research. Keim [Paul Keim, PhD, acting director of the NSABB and a microbial geneticist at Northern Arizona University] and many of the other presenters said scientists need to come up with a framework and guidelines themselves to avoid lawmakers stepping in to steer policies, which would likely be even more burdensome.

[Byline: Lisa Schnirring]

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[The rather scant information released so far indicates, not unexpectedly, that the mutations affecting transmissibility so far identified are all located in the HA genome subunit of the avian A(H5N1) influenza virus genome. The release of more precise information is still awaited. - Mod.CP]

Das Vogelgrippe-Virus könnte nach einer neuen Studie leicht zu einer weltweiten Gefahr werden. Demnach reichen nur fünf Mutationen im Erbgut des Erregers, damit sich dieser per Tröpfcheninfektion auch von Mensch zu Mensch verbreiten kann.

Wenige Mutationen könnten Vogelgrippe für Menschen gefährlich machen
22.06.2012 - Focus

Gruß
Birgitt

Das war schon vor einem Jahr mal in dne Medien. Aber jetzt hat man auch noch veroeffentlicht, wie das ablaufen soll. Super. Hilft bestimmt mal gegen die Ueberbevoelkerung.

AVIAN INFLUENZA, HUMAN (56): AIRBORNE SPREAD MUTATIONS
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[1]

Date: Fri 22 Jun 2012
Source: Science 22 June 2012: Vol. 336 no. 6088 pp. 1494-1497 (H5N1Special edition) [edited]
http://www.sciencemag.org/content/336/6088/1494.full


Public at Last, H5N1 Study Offers Insight Into Virus's Possible Path to Pandemic
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Depending on your point of view, the study that appears on page 1534 of this issue of Science marks another good week for public health experts trying to protect a vulnerable world from a new influenza pandemic -- or for future bioterrorists bent on unleashing one.

The paper, from a laboratory led by virologist Ron Fouchier of Erasmus MC in Rotterdam, the Netherlands, describes how a handful of mutations might give the H5N1 avian influenza virus, which typically infects birds, the potential to move easily between mammals and touch off a human flu pandemic. It appears after more than 8 months of often fierce international debate over whether the results should be made public -- and whether researchers should have conducted the experiments at all.

Late last year [2011], the U.S. National Science Advisory Board for Biosecurity (NSABB) unanimously asked Science not to publish the study's details. (The journal agreed, in principle.) But in March [2012], the same board voted 12 to 6 in favor of full publication after reviewing a revised and extended version of the manuscript and other evidence (Science, 6 Apr 2012, p. 19). Along the way, the debate prompted influenza scientists to self-impose a landmark moratorium on some types of H5N1 research (see p. 1496), the U.S. government to set new controls on taxpayer-funded studies involving potentially dangerous pathogens, and the Dutch government to consider blocking publication by invoking export-control laws.

The paper is the 2nd one in 2 months to suggest that H5N1 has pandemic potential. Last month, Nature published a similar study by Yoshihiro Kawaoka of the University of Wisconsin, Madison, and the University of Tokyo was also caught up in the controversy (Science, 4 May, p. 529).

Until now, Fouchier had publicly discussed his study in only very general terms, including in a talk at a September 2011 flu meeting in Malta that triggered wide media coverage. Seeing the data in full is "sobering," says influenza expert Nancy Cox of the U.S. Centers for Disease Control and Prevention in Atlanta, because it suggests that it's easier for H5N1 to trigger a pandemic than other studies --including her own -- had indicated. In combination with the Kawaoka paper, Fouchier's findings shed light on how the virus could become pandemic, says Malik Peiris of the University of Hong Kong, and how public health officials might watch for mutations that could send it on its way.

Although it has decimated poultry flocks and killed more than 600 people since it 1st surfaced in 1997, H5N1 has not touched off a pandemic in humans because it hardly ever spreads from one person to the next -- and some scientists think it never will. To become pandemic, the virus would have to become "airborne," or able to spread via tiny droplets spewed out during coughing or sneezing. That is how other influenza strains spread among humans, and both Fouchier and Kawaoka wanted to know which mutations might allow H5N1 to do the same.

There's a key difference between the studies, however. Kawaoka created a hybrid virus: He took the gene for a viral protein called hemagglutinin from an avian H5N1 strain and stitched it together with 7 other gene segments from the pandemic H1N1 virus that swept the world in 2009 and 2010, and which is already well-adapted to humans. From this starting point, it took just 4 mutations in the hemagglutinin gene to create a virus that could travel through the air from one infected ferret -- a popular animal model for human infection -- and infect another. But Kawaoka's hybrid has not yet been found in nature.

In contrast, "the strong point" of Fouchier's study, Cox says, is that it started out with an actual H5N1 virus isolated from a human victim in Indonesia. In an e-mail to Science, Kawaoka agreed that Fouchier's study addresses the most urgent question more directly. "Ron's data are very important," he said.

Fouchier's team 1st inserted several mutations they knew might help the virus adapt for mammalian spread. One key target was the virus's receptor binding site, the area within the hemagglutinin molecule that makes 1st contact with the host cell; scientists already knew that 2 mutations there can make the virus prefer mammalian cells over bird cells. Another mutation, in the polymerase protein complex, allows the virus to replicate in the cool environment of the human upper respiratory tract rather than in bird intestines, the much warmer environment where it usually resides.

These initial mutations alone didn't do the trick, however, so Fouchier's team decided to try a time-honored method to encourage a pathogen to adapt to a new host: They passed the virus from ferret to ferret by directly inoculating uninfected animals with nasal samples from infected ones and repeated the procedure a total of 10 times. (In his Malta talk, Fouchier called this a "really stupid" approach, a phrase widely interpreted to mean he regretted it. In fact, he says, he just meant that the technique, called passaging, is a simple one compared to the sophistication of creating targeted mutations. The confusion may have stemmed in part from the fact that the Dutch word for "stupid" can also mean "simple.")

The end result was a virus that could move through the air from one caged ferret to another right next to it; in a 1st experiment, the virus transmitted from cage to cage in 3 out of 4 instances.

Prior to publication, media reports suggested that airborne transmission required 5 mutations. The reality is more complex. Each of Fouchier's transmissible viruses had at least 9 mutations, 5 of which were shared by all. This core quintet may be sufficient, the team writes, but the big question is whether one or more of the other changes also plays an important role, Peiris says.

Fouchier already knows part of the answer. Once his team achieved transmission in the summer of 2011, the researchers began additional experiments to identify the minimum set of mutations needed to make the virus airborne. But before those experiments were finished, they submitted their manuscript to Science, worrying that Kawaoka or other scientists might beat them to the punch. "Usually when you discover something important, somebody else is discovering it, too," Fouchier says. (He was right: Kawaoka, who says he didn't know about Fouchier's work, had submitted his manuscript less than 2 weeks earlier.) Now, Fouchier declines to discuss the results from the additional experiments, which are on hold as a result of the moratorium.

The published paper shows that the core set of 5 mutations includes the 3 that the team introduced themselves and 2 more that arose during passaging. And the resemblance to what Kawaoka found is "quite remarkable," says James Paulson, a glycobiologist at the Scripps Research Institute in San Diego, California. Both teams found that 2 mutations at the receptor binding site -- one of them identical in the 2 studies -- are important; both discovered an additional mutation that makes hemagglutinin lose a sugar group, which apparently helps make room for the mammalian host cell receptor. Kawaoka also found a mutation in the hemagglutinin's stalk that improves the virus's stability and compensates for the other mutations, Paulson says. "It's tempting to think" that one of Fouchier's mutations plays a similar role -- although it's not in the stalk but in another area where 3 hemagglutinin molecules align to form a so-called trimer.

Cox says she's "surprised" that it didn't take more mutations. In a study with Paulson published online by Virology in November [2011], her team also mutated the receptor-binding site, but they had to make several other changes -- including slotting in a human-adapted version of another viral gene called neuraminidase -- to get airborne transmission. That led the authors to conclude that the virus might require "extensive evolution" to become pandemic. Fouchier's paper upsets that reassuring notion.

Both papers will aid surveillance efforts because they suggest which genetic changes to look out for in H5N1, Peiris says. But they also point to a limitation: Several mutations can have the same effect on the virus. "It wouldn't be appropriate to focus just on these mutations," Peiris says. "The virus has different ways to go from A to B." More research is needed to discover how many ways, he says.

In a 2nd paper published this week and co-authored by Kawaoka and Fouchier (p. 1541), a group led by mathematician Derek Smith of the University of Cambridge in the United Kingdom takes a stab at understanding the likelihood of the emergence of a pandemic H5N1 strain. The researchers 1st combed through surveillance databases to determine whether the mutations identified in the 2 controversial studies have already appeared in nature. They found that many H5N1 isolates are 3 -- and in a few rare cases, just 2 -- mutations away from Kawaoka's quartet and 4 from Fouchier's quintet.

They then developed a model of viral evolution to test whether existing viruses, once they happen to infect a mammalian host, might accumulate the missing mutations and become excreted in respiratory droplets, which could start a chain of transmission. The model takes into account a variety of factors, such as the duration of the infection and whether individual mutations by themselves benefit the virus. The conclusion, says 1st author Colin Russell of the University of Cambridge, is that a virus that is only 3 mutations away from the full set is "likely" to acquire them and end up in droplets. But the paper can't put a number on that risk; there are too many unknowns.

"You can do a lot of fancy maths, but in the end the probability is hard to pin down," Peiris says. Still, "it's a model of how modeling should be done," says Steven Wolinsky, who studies HIV evolution at Northwestern University in Chicago, Illinois. "They do a very nice job of explaining all the caveats."

The study was presented both at a World Health Organization meeting about the papers in February [2012] and during the 2nd NSABB review in March [2012], and it helped convince a majority on the panel that H5N1's risks were real enough to warrant publishing Fouchier's paper, says NSABB acting chair Paul Keim, a microbial geneticist at Northern Arizona University in Flagstaff.

Another factor that swayed the board to support publication, NSABB members say, is that the published version of Fouchier's paper does a much better job of clarifying the lethality of his airborne virus than the 1st version they read. Fouchier says the draft he submitted to Science did not discuss whether his airborne viruses killed the ferrets they infected. But 2 of the 3 reviewers asked for additional experiments examining lethality, which Fouchier said took just a week to perform, "so we did them," and added a line to the paper describing the outcome.

That language -- combined with Fouchier's remarks in interviews and sometimes hyperbolic press coverage -- appeared to suggest that the airborne mutants were extremely lethal, which "greatly alarmed" many NSABB reviewers, says virologist Robert Webster of St. Jude Children's Research Hospital in Memphis, Tennessee, an influenza expert who was asked to advise NSABB on the 2 papers. In fact, however, none of the ferrets had died from airborne transmission; 6 ferrets that had the virus squirted directly into their trachea all died. But that outcome is "not very relevant" for evaluating the virus's risk, Fouchier notes, because that's not how humans or animals typically contract flu. He says the manuscript NSABB saw in its 1st review last year [2011] made clear that the different routes of infection led to different outcomes.

But Webster says the results were presented in a way that confused NSABB. He says that the experienced virologists involved in NSABB's discussions -- including himself -- should have pushed harder to clarify those results and emphasize that lethality in ferrets does not necessarily predict lethality in humans. When the lethality data finally became clearer in the months after the NSABB's initial recommendation, many members say they began to reconsider. In retrospect, Fouchier says, "we should have ignored [Science's] reviewers' request for lethality data," given the confusion that ensued.

The publication of Fouchier's paper isn't likely to be the last word on such issues, however, especially as influenza researchers seek to restart similar studies now stalled by the moratorium. Many, however, are treading cautiously, eager to avoid replaying the drama of the last 8 months. Fouchier, for his part, says he's "sick of all these discussions," and he declined to release his 1st manuscript to reporters in order to help clarify how the story unfolded, a step Keim says he would support. "I want to move on," Fouchier says. "Maybe in 5 or 10 years' time, when someone writes a book about all of this."

[Byline: Martin Enserink with reporting by David Malakoff]

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[2]
Date: Thu 21 Jun 2012
Source: CIDRAP Newa [abbrev., edited]
http://www.cidrap.umn.edu/cidrap/conten ... chier.html


Fouchier study reveals changes enabling airborne spread of H5N1
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A study showing that it takes as few as 5 mutations to turn the H5N1 avian influenza virus into an airborne spreader in mammals -- and that launched a historic debate on scientific accountability and transparency -- was released today in Science, spilling the full experimental details that many experts had sought to suppress out of concern that publishing them could lead to the unleashing of a dangerous virus.

In the lengthy report, Ron Fouchier, PhD, of Erasmus Medical Center in the Netherlands and colleagues describe how they used a combination of genetic engineering and serial infection of ferrets to create a mutant H5N1 virus that can spread among ferrets without direct contact. They say their findings show that H5N1 viruses have the potential to evolve in mammals to gain airborne transmissibility, without having to mix with other flu viruses in intermediate hosts such as pigs, and thus pose a risk of launching a pandemic.

The Fouchier study is paired with another in which an international team used mathematical modeling to reach the conclusion that, given how few mutations it took to yield an airborne transmissible virus, such a virus probably could evolve in nature, though it's not possible to quantify the risk. They noted that 2 of the 5 mutations in the airborne virus already are common in natural H5N1 viruses. "We now know that we're living on a fault line," said Derek J. Smith, PhD, of the University of Cambridge, senior author of the modeling study, at a Science press conference yesterday. "It could do something. What we need to know is how likely is that."

Fouchier's study is one of 2 H5N1 transmissibility experiments that sparked a fierce controversy beginning late last fall, and is the 2nd to see print. The 1st study, led by Yoshihiro Kawaoka, DVM, PhD, of the University of Wisconsin and the University of Tokyo, was published in Nature in early May. Kawaoka's team used a somewhat different approach to achieve the same goal as Fouchier. Kawaoka and colleagues introduced mutations in the hemagglutinin (HA) gene from an H5N1 virus and then combined it with 7 genes from a 2009 H1N1 virus, creating a hybrid virus that was able to spread by air between ferrets. They said the study demonstrated that a flu virus with H5 HA can spread among mammals -- something that natural H5N1 viruses have done extremely rarely, if at all.

When word of the Fouchier and Kawaoka studies got out last fall, it sparked concern about the potential for creation and intentional or unintentional release of a pandemic virus. This led to a review by the US National Science Advisory Board for Biosecurity (NSABB), which advises the Department of Health and Human Services. After long deliberations, the board recommended in December [2011] that both studies be stripped of key details before publication, but that the details be shared with selected scientists and officials on a need-to-know basis. But after the authors -- especially Fouchier -- provided some additional information and clarifications, the board recommended on 30 Mar 2012 that the full versions of both studies be published, and federal health officials endorsed the recommendation. The NSABB voted unanimously in favor of publication for Kawaoka's paper, but it split 12-6 on Fouchier's.

The heated controversy over publication of the studies prompted a group of 39 leading flu researchers, including Fouchier and Kawaoka, to impose a voluntary moratorium on research designed to increase the transmissibility of H5N1 viruses in mammals. The moratorium remains in effect, awaiting the further development of government policies on biosafety and biosecurity for such research. The controversy also prompted the US government in March [2012] to outline a new general policy on the handling of "dual use research of concern," or DURC, meaning research that could be used for either good or ill. US officials said this week that further details on that policy will be released soon in the form of draft guidance for institutional biosafety committees.

In a question-and-answer sheet released with Fouchier's study, Erasmus officials said the paper was revised to better explain the research goals and the benefits to public health, and "display items" were added to explain the virulence and transmission properties of the airborne virus, but the methods and results were not changed. Also, the manuscript was revised to improve clarity, since it was expected to draw a broader readership than such reports usually do. As for safety, the experiments were conducted in an Animal Biosafety Level 3 (ABSL3+) lab at Erasmus, the report says. Inspectors from the Dutch government and the US Centers for Disease Control and Prevention approved the facilities and procedures before and during the research. Researchers wore protective equipment and were offered seasonal and H5N1 flu vaccines.

Fouchier's team started with an H5N1 virus collected in Indonesia and used reverse genetics to introduce mutations that have been shown in previous research to make H5N1 viruses more human-like in how they bind to airway cells or in other ways. Avian flu viruses prefer to bind to alpha2,3-linked sialic acid receptors on cells, whereas human flu viruses prefer alpha2,6-linked receptors. In both humans and ferrets, alpha2,6 receptors are predominant in the upper respiratory tract, while alpha 2,6 receptors are found mainly in the lower respiratory tract. The amino acid changes the team chose included N182K, Q222L, and G224S, the numbers referring to positions in the virus's HA protein, the viral surface molecule that attaches to host cells. Q222L and G224S together change the binding preference of H2 and H3 subtype flu viruses, changes that contributed to the 1957 and 1968 flu pandemics, according to the report. And N182K was found in a human H5N1 case.

The scientists created 3 mutant H5N1 virus strains to launch their experiment: one containing N182K, one with Q222L and G2242, and one with all 3 changes, the report explains. They then launched their lengthy series of ferret experiments by inoculating groups of 6 ferrets with one of these 3 mutants or the wild-type H5N1 virus. Analysis of samples during the 7-day experiment showed that ferrets infected with the wild-type virus shed far more virus than those infected with the mutants.

In a 2nd step, the team used a mutation in a different viral gene, PB2, the polymerase complex protein. The mutation E627K in PB2 is linked to the acquisition by avian flu viruses of the ability to grow in the human respiratory tract, which is cooler than the intestinal tract of birds, where the viruses usually reside, according to the report. The researchers found that this mutation, when added to 2 of the HA mutations (Q224L and G224S), did not produce a virus that grew more vigorously in ferrets, and the virus did not spread through the air from infected ferrets to uninfected ones. Seeing that the this mutant failed to achieve airborne transmission, the researchers decided to "passage" this strain through a series of ferrets in an effort to force it to adapt to the mammalian respiratory tract -- the move that Fouchier called "really, really stupid," according to a report of his initial description of the research at a European meeting last September [2011]. They inoculated one ferret with the 3-mutation strain and another with the wild-type virus and took daily samples until they euthanized the animals on day 4 and took tissue samples (nasal turbinates and lungs). Material from the tissue samples was then used to inoculate another pair of ferrets, and this step was carried out 6 times. For the last 4 passages, the scientists used nasal-wash samples instead of tissue samples, in an effort to harvest viruses that were secreted from the upper respiratory tract.

The amount of mutant virus found in the nasal turbinate and nose swab samples increased with the number of passages, signaling that the virus was increasing its capacity to grow in the ferret upper airway. In contrast, viral titers in the samples from ferrets infected with the wild-type virus stayed the same. The next step was to test whether the viruses produced through passaging could achieve airborne transmission. 4 ferrets were inoculated with samples of the "passage-10" mutant virus, and 2 ferrets were inoculated with the passage-10 wild strain. Uninfected ferrets were placed in cages next to the infected ones but not close enough for direct contact.

The ferrets exposed to those with the wild virus remained uninfected, but 3 of the 4 ferrets placed near those harboring the mutant virus did get infected, the researchers found. Further, they took a sample from one of the "recipient" ferrets and used it to inoculate another ferret, which then transmitted the virus to 2 more ferrets that were placed near it. Thus, a total of 6 ferrets became infected with the mutant virus via airborne transmission. However, the level of viral shedding indicated the airborne virus didn't transmit as efficiently as the 2009 H1N1 virus does.

In the course of the airborne transmission experiments, the ferrets showed signs of illness, including lethargy, loss of appetite, and ruffled fur. One of the directly inoculated ferrets died, but all those infected via airborne viruses survived. When the scientists sequenced the genomes of the viruses that spread through the air, they found only 2 amino acid switches, both in HA, that occurred in all 6 viruses: H103Y and T156A. They noted several other mutations, but none that occurred in all 6 airborne viruses.

"Together, these results suggest that as few as 5 amino acid substitutions (4 in HA and one in PB2) may be sufficient to confer airborne transmission of [highly pathogenic avian flu] H5N1 virus," the researchers wrote. In further steps, the researchers inoculated 6 ferrets with high doses of the airborne-transmissible virus; after 3 days, the ferrets were either dead or "moribund." "Intratracheal inoculations at such high doses do not represent the natural route of infection and are generally used only to test the ability of viruses to cause pneumonia," the report notes.

In addition, they assessed whether an existing H5N1 vaccine would be likely to offer any protection against the virus. They generated a hybrid virus consisting of the mutant HA and PB2 genes combined with 6 genes from a mouse-adapted virus called PR8. This hybrid reacted well with antibodies generated by several existing H5N1 vaccines, suggesting that the vaccines could provide some protection. Finally, the team tested whether antibodies in elderly people (over 70) could recognize the PR8/H5 hybrid virus. The answer was no, suggesting they would have little immunity to it or to the airborne-transmissible mutant.

The scientists note that substitutions Q222L and G224S, which are linked to changes in binding preference, have been found in avian H2 and H3 viruses in nature, though not in H5N1 viruses. The other 3 mutations that were found consistently in the airborne-transmissible mutant have all been found in natural H5N1 viruses, which suggests that they don't impair viral "fitness." H103Y has been identified only once, but E627K in PB2 has been found in about 27 percent of avian H5N1 sequences and about 29 percent of human H5N1 sequences, the report says. Also, Q222L is one of the 4 key mutations identified in the Kawaoka study.

"Given the large numbers of HPAI A/H5N1 virus-infected hosts globally, the high viral mutation rate, and the apparent lack of detrimental effects on fitness of the mutations that confer airborne transmission, it may simply be a matter of chance and time before a human-to-human transmissible A/H5N1 virus emerges," the scientists state. In the accompanying modeling study, Cambridge researchers sought to estimate the risk that an airborne-transmissible virus like Fouchier's could evolve naturally. Analyzing surveillance data for the past 15 years, they noted that 2 of the mutations involved (T156A and E627K) are common in natural H5N1 isolates, and a number of isolates had both changes. The researchers developed a mathematical model of how viruses replicate and evolve in a mammal and assessed the chances that the 3 additional needed mutations could evolve in a single host or a short chain of transmission. Among the factors they included were random mutation, prolonged infections, transmission, immune response, and deleterious mutations. "With the information we have, it is impossible to say what the exact risk is of the virus becoming airborne transmissible among humans," senior author Smith said in a Cambridge press release. "However, the results suggest that the remaining 3 mutations could evolve in a single human host, making a virus evolving in nature a potentially serious threat."

Early indications are that the release of Fouchier's study will not end the controversy over its publication and the broader problem of dealing with dual-use research. Some biosecurity experts, though, voiced misgivings today. Eric S. Toner, MD, senior associate in the Center for Biosecurity at the University of Pittsburgh Medical Center, commented by e-mail, "I think that the publication does increase the probability that others will want to replicate the experiments and pursue this line of research further. The more labs that are doing this research, the more likely an accidental escape becomes. "Clearly if all the lab workers were effectively vaccinated against the virus, the risk of accidental escape would decrease. The paper says the lab workers were offered H5N1 vaccination. It would be nice to know if it was required and that the workers had good antibody titers as well as that the specific H5N1 vaccine had demonstrated efficacy against the research strain." Toner also said the findings strongly support the concern that a naturally occurring H5N1 pandemic is possible. But he called the findings "only modestly useful" for flu surveillance.

Michael T. Osterholm, PhD, MPH, director of the University of Minnesota's Center for Infectious Disease Research and Policy, publisher of CIDRAP News, said he supports the type of research done by Fouchier and Kawaoka and thinks it should continue, but not in labs all over the world. He said the Fouchier and Kawaoka studies mark the 1st time humans have ever broken the barrier between nontransmissibility and transmissibility of a pathogen in animals. The heart of the issue is transmissibility, not the virulence of the lab-derived viruses, he asserted. What a ferret-transmissible H5N1 virus would do in humans is unknown, he commented. "But if it started to circulate, such as in swine, all bets are off. Once you break that transmissibility barrier, this virus is open for serious mischief." The details of Fouchier's study "should've been disseminated on a need-to-know basis," Osterholm said. "I think we punted on this, we didn't exhaust every possibility for disseminating it."

He was referring to the conclusion of most officials and scientists involved that it was not possible to quickly devise a way to share the details only with a select group. "We're making it much easier for everyone to do this," he said. "What if some vaccine manufacturing company in a developing country decides they want to work with this and it gets out?" He commented that the release of a dangerous flu virus through a lab accident has already happened once, with the re-emergence of the H1N1 virus in 1977: "That was a clear case of a virus that leaked out of work that the Russians were doing." Osterholm also warned that the issues raised by the Fouchier paper won't go away, saying research papers now in the pipeline will raise even greater concerns about possible misuse and biosafety. "I think the federal government and the NSABB are very poorly position to deal with the future manuscripts that are already in the pipeline," he said.

Another NSABB member, David A. Relman, MD, of Stanford University, expressed the view that the Fouchier study shouldn't have been done in the 1st place, let alone published. "Basically, I was and still am opposed to this particular work, because the risks outweighed the benefits, from the start, and still do," he commented by e-mail. "I don't believe that the benefits, even if they (e.g., early detection in birds and animals) could be realized in the near term, are worth the significant risks. Accidental release is a major concern, as well as malignant disregard for public safety. "We already know enough to push full out for a broadly protective H5 vaccine. What more do we need? Do we really want to wait for a mammalian respiratory transmissible H5 to show up in nature? The general public appears to be largely opposed or at least highly concerned. A real moratorium should continue for the foreseeable future."

A more positive view of the research was offered by Angus Nicoll, CBE, head of the Influenza Program at the European Centre for Disease Prevention and Control. He said it's significant that the 2 different experiments by Fouchier and Kawaoka produced viruses with some degree of airborne transmissibility, which "suggests a somewhat greater risk of this happening in nature than if you found there was only one way and one virus that got you to this stage. However it must be remembered that these viruses are only just about transmissible in ferrets (a comment made by Fouchier in Washington was that these viruses really did not spread very well naturally, and their pathogenicity in the ferrets seems to be lower than wild A(H5N1)." Nicoll added that much more study of the viruses is needed: "Can the results be replicated by others? For example are they still pathogenic in poultry? That is why the moratorium on research is unfortunate though it's quite understandable given the strong feelings and concerns. I hope there will be some agreement in the autumn following a wider meeting organized by WHO [World Health Organization] that allow it to restart under the necessary level of containment." He also commented that the findings are valuable for identifying which mutations in nature are most worrisome and for guiding the development of H5N1 diagnostic tests and vaccines.

References:
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Herfst S, Schrauwen EJA, Linster M, et al. Airborne transmission of influzna A/H5N1 virus between ferrets. Science 2012 Jun 22;336 (early online publication) [Full text]

Russell CA, Fonville JM, Brown AEX, et al. The potential for respiratory droplet-transmissible A/H5N1 infuenza virus to evolve in a mammalian host. Science 2012 Jun 22;336 (early online publication) [Full text]

[Byline: Robert Roos]

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[The team from Cambridge University have looked to see whether such mutations could emerge naturally and if so its likelihood. They studied the genetic structure of 3000 bird viruses and 400 that occur in humans. They found some of these viruses had 2 of the key changes needed to become airborne. Mathematical modelling suggested it was indeed possible for a virus to develop the 3 further changes required during the course of an epidemic. It is the 1st time it has been shown that it is possible for bird flu to become airborne, but the research team was unable to determine precisely how likely this was to happen.

Prof. Derek Smith, who led the analysis, said more information was needed. He said researchers required a better understanding of how flu viruses were transmitted between people in order to develop a clearer idea of the likelihood of the emergence of an airborne strain of bird flu. (From: BBC-News http://www.bbc.co.uk/news/science-environment-18534676). - Mod.CP]

Inzwischen ist bekannt, dass das tödliche Vogelgrippevirus eine menschliche Pandemie verursachen kann. Darum ist es umso dringender, die verbleibenden offenen Fragen zu klären. Die Biologie des H5N1-Vogelgrippevirus steckt voller Widersprüche. Das Virus ist weit verbreitet, aber schwer zu entdecken. Es tötet mehr als die Hälfte der Menschen, bei denen eine Infektion entdeckt wurde, aber Tausende, die ihm ausgesetzt waren, zeigen keinerlei Beschwerden. Es scheint nur wenige Mutationsschritte von der Fähigkeit entfernt, sich von Mensch zu Mensch zu übertragen, und doch hat es diese Schritte in über 16 Jahren rascher Entwicklung nicht vollzogen. Diesen Juni deckten Wissenschaftler die Mutationen auf, welche H5N1 befähigen, sich durch die Luft von einem Frettchen zum anderen auszubreiten ...

Fünf Fragen zu H5N1
25.09.2012 - spektrum

Gruß
Birgitt