Avian Influenza MARCH Lab Report: Scientific Developments In The Fight Against H5N1

A couple of months ago, H5N1 was only found in a few countries in SE Asia. In the last month alone H5N1 has spread to Africa and most of Europe. As quickly as BF is spreading, the scientific knowledge is growing. At the end of last year, it was discovered that the 1918 Spanish Flu was an Avian Influenza. Just this week it's been reported that scientists have had a major breakthrough is the very mechanics of how the virus becomes human transmissable.

The intent of this thread will be to focus on the scientific front in our battle with H5N1.

It seems appropriate to inaugrurate this thread with some photos of H5N1... first published last November:

http://www.timebomb2000.com/vb/showthread.php?t=175262
First pictures of H5N1 published
From: Agence France-Presse From correspondents in Stockholm
November 07, 2005

THE first high-resolution close-up photographs of the H5N1 avian flu virus to be taken by a scanning electro microscope appeared in the Swedish daily Dagens Nyeter (DN) overnight.

Click to expand...

Scientists dissecting bird flu virus mutations
Test can identify changes that could cause a pandemic​

Reuters
Updated: 2:24 p.m. ET March 16, 2006

WASHINGTON - Scientists said on Thursday they had identified some of the mutations the H5N1 avian influenza virus needs to gain a permanent foothold in the human population, causing a greatly feared pandemic.

They said the test they used, called a glycan microarray, might be useful in monitoring the virus in birds and as it infects people, to see if it is mutating into a form that would allow it to pass easily from person to person.

H5N1 has moved steadily across Asia and into Europe since it reappeared in 2003, and has picked up speed in recent weeks. It has killed just over 100 people but remains mostly a virus of birds.

No one can predict when, or even if, it will evolve into a form that transmits easily from one person to another, but fears are that it will. Scientists have been examining the virus when they can get samples and trying to predict just which changes are needed to make it change from a bird-specific to a human-specific form.

Ian Wilson and a team at The Scripps Research Institute in La Jolla, California looked at a structure on the surface of all influenza viruses called hemagglutinin. It is the “H” in H5N1 and there are 16 known types of hemagglutinin.

Only three -- H1, H2 and H3 -- have been known to cause human disease and they caused the last three great influenza pandemics, in 1918, 1957 and 1968.

“When pandemics start, we really don’t know, with the first virus that enters the human population, how well it is adapted to humans,” Wilson said in a telephone interview.

Resembling pandemic virus
Working with flu experts Terence Tumpey of the Centers for Disease Control and Prevention in Atlanta and Jeffery Taubenberger of the Armed Forces Institute of Pathology in Rockville, Maryland, Wilson’s team dissected and imaged a sample of influenza virus that killed a 10-year-old Vietnamese boy in 2004.

They found its hemagglutinin looked very similar to the virus that caused the 1918 pandemic, which killed anywhere between 50 million and 100 million people. It looked less similar to H5N1 taken from a duck in Singapore.

“Of the H5N1 viral isolates studied to date, A/Vietnam/1203/2004 (Viet04) is among the most pathogenic in mammalian models, such as ferrets and mice,” the researchers wrote in their report, published in the journal Science.

Hemagglutinin allows the virus to attach to the cells it infects, and it has to be able to grapple a structure called sialic acid, which is slightly different in different animal species.

So to pass from birds to humans, the virus must mutate enough to allow it to bind to, for instance, both a human lung cell and a chicken’s intestinal cell.

Not all or nothing
Wilson’s team identified a structure that, in the human configuration, is called an alpha 2-6 receptor and in birds an alpha 2-3 receptor. It must change from the bird to human configuration, they found, to cause human epidemics.

This “critical step ... appears to be one of the reasons why most avian influenza viruses, including current avian H5 strains, are not easily transmitted from human-to-human following avian to human infection,” they wrote.

Earlier studies had shown it took only two changes to make the 1918 virus look just like a purely avian virus. That may suggest it may not take much mutation to change a bird virus into a human pandemic strain.

The test may help monitor for these changes, Wilson said.

“This test that you can do for receptor testing specificity, this glycan array, is something you could possibly think about using in the field,” Wilson said.

So if the virus has not mutated yet, why does it ever infect people? “It’s not an all or nothing,” Wilson said.

“It’s a preference. At higher doses, doses that normally you and I wouldn’t pass the virus on to one another... you can overcome this (species) barrier.”
Copyright 2006 Reuters Limited. All rights reserved. Republication or redistribution of Reuters content is expressly prohibited without the prior written consent of Reuters.

URL: http://www.msnbc.msn.com/id/11857243/

Sugar Molecules & Cell Membranes... a key

Bird flu mutation created in lab​

STACEY SINGER
COX NEWS SERVICE

WEST PALM BEACH, Fla. - Scientists at The Scripps Research Institute have broken new ground in the effort to track the evolution of bird flu, developing a technology that offers an early warning system for the flu's feared transition from bird slayer to human menace.

It's a rare but deadly event for bird flu to jump the species barrier. So rare, that scientists are aware of only a few previous occurrences: The first was the Spanish flu of 1918. It killed an estimated 30 million to 50 million people worldwide.

The second, the Asian flu, hit in 1957, killing 4 million. The third, in 1968, was the Hong Kong flu, which left 37,000 dead in the United States.

The new bird flu strain sweeping the globe - labeled H5N1 - already has infected 177 people and killed 98 of them. And yet, scientists say, it's not very contagious. For now, H5N1 favors the cells found in birds' intestinal tracts.

If and when its preference changes to people, health officials fear a cascade of human-to-human infection will follow, leaving millions dead. Congress in December appropriated $3.8 billion to prepare for and prevent that day.

In a paper released Thursday by the journal Science, the team based at Scripps' La Jolla, Calif., headquarters said they had created mutations in the lab that would likely increase bird flu's attraction to human cells

The potentially deadly mutation was identified with new technology called a glycan microarray. Developed at Scripps as part of a large international collaborative effort, the technology allows scientists to rapidly spot mutations in a key viral protein..

The Science paper suggests the technology may have an important role to play in mapping out whether bird flu is evolving into a greater threat to humans.

Molecular biologist James Stevens said this type of research can provide health authorities with an early-warning system to look for particular mutations that might lead to human adaptation.

"We can plan ahead if we know what we think might happen," Stevens said.
Making a dangerous virus more dangerous might sound counter-intuitive, but the Scripps scientists were working with just a small piece of influenza - one that's incapable of causing infection on its own. It came from a strain of H5N1 that killed a 10-year-old boy in Vietnam in 2004.

"The more these viruses interact with the human population, the more likely it is that it could adopt this type of mutation," Stevens said.

The scientists focused on one protein that studs the influenza virus exterior, called hemagglutinin. It's the "H" in H5N1, and it's critical because its job is to recognize and latch on to cells. Viruses cannot replicate on their own - they must hijack healthy cells to do it for them.

Viruses find their cellular victims by looking for the distinctive sugar molecules that coat cells' outer membranes.

The authors of the Science paper were able to change the H5N1 hemagglutinin enough for it to latch more easily onto sugars found on the exterior of cells lining human lungs.

Encouragingly, Stevens said, the mutations' attachment to the cells was weak - suggesting the existing virus cannot easily cross the species barrier outside a lab.

"It's surprising that it wasn't easy to convert," Stevens said. "There may be other factors that prevent it. That's not to say that Mother Nature won't come along and throw us a curve ball."

Dr. Robert Belshe, a physician at Saint Louis University's Infectious Diseases and Immunology department, said this research and others gives him hope that there's still time to head off a human pandemic. Belshe is conducting clinical trials on investigational avian flu vaccines.

The current avian flu outbreak was first recognized nine years ago. As it spreads in birds, one of the signs to watch for will be the mutation described in the Science paper, he said.

"If that occurred naturally it would be a very alarming event," Belshe said.

Vaccines and drugs offer the best defense, but it takes time and money to prepare the right attack and then make adequate quantities. Knowing what to watch for will help buy more time, Belshe said.

Scientists at Scripps have traveled this road before. Stevens recently analyzed mutations believed to convert the 1918 flu to a human strain. Twenty years earlier, Scripps' James Paulson helped develop a method to see how flu bound to sugars on cell membranes. It enabled a team including Wilson to find the amino acid combination that cause Hong Kong flu to bind to human cells.

Scripps' Ian Wilson also played a key role in understanding the Hong Kong flu. Working in the lab of Harvard's Don Wiley, he helped characterize the shape of hemagglutinin in 1981. In La Jolla, in the atrium of the Skaggs Institute for Chemical Biology at Scripps, a 3-foot-tall, three-dimensional model of a hemagglutinin molecule sits on a pedestal

When Scripps President Richard Lerner managed to recruit Wilson away from Harvard, he marked the occasion by commissioning the enormous model, Wilson said.

That model happens to be of the hemagglutinin found on the deadly Hong Kong flu. Coincidentally, its the source for the mutation that Stevens found gave H5N1 the strongest potential foothold on human cells.

Will a similar change happen in nature?

"It's all down to statistics and chance," Stevens said. "It could happen tomorrow, it could happen ten years from now, it may never happen."

In addition to Stevens, Wilson and Paulson, the Science paper was authored by Terrence Tumpey of the Centers for Disease Control and Prevention's Influenza Branch, Jeffery Taubenberger of the Armed Forces Institute of Pathology and Scripps molecular biologist Ola Blixt.

Blixt, working with Paulson and the Consortium for Functional Glycomics, designed the glycan microarray that was used to assess how well the bird flu hemagglutinin attached to sugars on the surface of human lung cells.

It's a clever technology, one that grew out of similar automation created for genetic screening.

Glass plates are covered with the ends of sugar molecules found on key cell types - 300 per plate. Fluorescent tagged hemagglutinin is washed across the plate. The spots that light up most brightly are the ones most able to bind the viral protein.

The science of glycomics, and the technology of glycan microarrays, is relatively young, but Paulson predicts it will lead to many more discoveries. Twenty years after he discovered why Hong Kong flu made the leap to humans, Paulson said, "The questions are the same, but the tools are a lot more sophisticated."

http://www.bellinghamherald.com/app...3170340&SectionCat=BUSINESS&Template=printart

Minor Mutations in Avian Flu Virus Increase Chances of Human Infection​

By Eric Sauter

Scientists at The Scripps Research Institute, the Centers for Disease Control, and the Armed Forces Institute of Pathology have identified what the researchers described as a possible pathway for a particularly virulent strain of the avian flu virus H5N1 "to gain a foothold in the human population."

The H5N1 avian influenza virus, commonly known as "bird flu," is a highly contagious and deadly disease in poultry. So far, its spread to humans has been limited, with 177 documented severe infections, and nearly 100 deaths in Indonesia, Vietnam, Thailand, Cambodia, China, Iraq, and Turkey as of March 14, 2006, according to the World Health Organization.

"With continued outbreaks of the H5N1 virus in poultry and wild birds, further human cases are likely," said Ian Wilson, Scripps Research professor of molecular biology and head of the laboratory that conducted the recent study. "The potential for the emergence of a human-adapted H5 virus, either by re-assortment or mutation, is a clear threat to public health worldwide."

Of the H5N1 strains isolated to date, the researchers looked at A/Vietnam/1203/2004 (Viet04), one of the most pathogenic H5N1 viruses studied so far. The virus was originally isolated from a 10-year-old Vietnamese boy who died from the infection in 2004. The hemagglutinin (HA) structure from the Viet04 virus was found to be closely related to the 1918 virus HA, which caused some 50 million deaths worldwide.

Using a recently developed microarray technology—hundreds of microscopic assay sites on a single small surface—the study showed that relatively small mutations can result in switching the binding site preference of the avian virus from receptors in the intestinal tract of birds to the respiratory tract of humans.These mutations, the study noted, were already "known in [some human influenza] viruses to increase binding for these receptors."

The study was published on March 16, 2006 by ScienceXpress, the advance online version of the journal Science.

Receptor specificity for the influenza virus is controlled by the glycoprotein hemagglutinin (HA) on the virus surface. These viral HAs bind to host cell receptors containing complex glycans—carbohydrates—that in turn contain terminal sialic acids. Avian viruses prefer binding to a2-3-linked sialic acids on receptors of intestinal epithelial cells, while human viruses are usually specific for the a2-6 linkage on epithelial cells of the lungs and upper respiratory tract. Such interactions allow the virus membrane to fuse with the membrane of the host cell so that viral genetic material can be transferred to the cell.

The switch from a2-3 to a2-6 receptor specificity is a critical step in the adaptation of avian viruses to a human host and appears to be one of the reasons why most avian influenza viruses, including current avian H5 strains, are not easily transmitted from human-to-human following avian-to-human infection. However, the report did suggest that "once a foothold in a new host species is made, the virus HA can optimize its specificity to the new host."

"Our recombinant approach to the structural analysis of the Viet04 virus showed that when we inserted HA mutations that had already been shown to shift receptor preference in H3 HAs to the human respiratory tract, the mutations increased receptor preference of the Viet04 HA towards specific human glycans that could serve as receptors on lung epithelial cells," Wilson said. "The effect of these mutations on the Viet04 HA increases the likelihood of binding to and infection of susceptible epithelial cells."

The study was careful to note that these results reveal only one possible route for virus adaptation. The study concluded that other, as yet "unidentified mutations" could emerge, allowing the avian virus to switch receptor specificity and make the jump to human-to-human transmission.

The glycan microarray technology, which was used to identify the mutations that could enable adaptation of H5N1 into the human population in the laboratory, could also be used to help identify new active virus strains in the field by monitoring changes in the receptor binding preference profile where infection is active, according to Jeremy M. Berg, the director of the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health (NIH). The glycan microarray was developed by The Consortium for Functional Glycomics, an international group led by Scripps Research scientists and supported by the NIGMS.

"This technology allows researchers to assay hundreds of carbohydrate varieties in a single experiment," Berg said. "The glycan microarray offers a detailed picture of viral receptor specificity that can be used to map the evolution of new human pathogenic strains, such as the H5N1 avian influenza, and could prove invaluable in the early identification of emerging viruses that could cause new epidemics."

Other authors of the study include James Stevens of Scripps Research; Ola Blixt of Scripps Research and Glycan Array Synthesis Core-D, Consortium for Functional Glycomics; Terrence M. Tumpey, Influenza Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention; Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, and; James C. Paulson, Scripps Research and Glycan Array Synthesis Core-D, Consortium for Functional Glycomics.

The work was supported by the National Institute of Allergy and Infectious Disease, the National Institute of General Medical Sciences and the National Institutes of Health.

http://www.scripps.edu/newsandviews/e_20060320/avian.html

Bird flu splits into two strains​

Last Update: Tuesday, March 21, 2006. 9:31am (AEDT)

The H5N1 bird flu in humans has evolved into two separate strains, a development that will complicate the search for a vaccine and the prevention of a pandemic, US researchers have reported.

The genetic diversification of the pool of H5N1 avian influenza viruses with the potential to cause a human influenza pandemic heightens the need for careful surveillance, researchers said at the International Conference on Emerging Infectious Diseases in Atlanta.

"Back in 2003 we only had one genetically distinct population of H5N1 with the potential to cause a human pandemic. Now we have two," said Rebecca Garten of the US Centres for Disease Control and Prevention, who helped conduct the study.

One of the two strains, or clades, made people sick in Vietnam, Cambodia and Thailand in 2003 and 2004 and the second, a cousin of the first, caused the disease in people in Indonesia in 2005.

Two clades may share the same ancestor but are genetically distinct - as are different clades, or strains, of the AIDS virus, the team from the CDC found.

"This does complicate vaccine development. But we are moving very swiftly to develop vaccines against this new group of viruses," said Dr Nancy Cox, chief of the CDC's influenza branch.

The H5N1 strain of bird flu has spread across Europe, Africa and parts of Asia and killed nearly 100 people worldwide and infected about 180 since it re-emerged in 2003.

Although it is difficult to catch bird flu, people can become infected if they come into close contact with infected birds.

Scientists fear the virus could mutate into a form that could pass easily between humans, triggering a pandemic in which millions could die.

All influenza viruses mutate easily, and H5N1 appears to be no exception.

But Dr Cox said the evolution of a second clade does not move the virus closer to human-to-human transmission.

"Like the group one or clade one viruses, the group two or clade two viruses are not easily transmitted from person to person," she said.

"It really doesn't take us closer to a pandemic. It simply makes preparing for the pandemic a bit more difficult."

The US Health and Human Services Department has already recognised the two strains and approved the development of a second H5N1 vaccine based on the second clade.

Several companies are working on H5N1 vaccines experimentally, although current formulations are not expected to protect very well, if at all, against any pandemic strain.

A vaccine against a pandemic flu strain would have to be formulated using the actual virus passing from person to person.

Researchers said while vaccines were needed against different strains of the virus, a vaccination against one clade could provide partial protection against another.

"We would expect the priming (of a patient) with a clade one (vaccine) could potentially reduce the severity of disease," Dr Cox said.

For their study, Ms Garten and colleagues analysed more than 300 H5N1 virus samples taken from both infected birds and people from 2003 through the summer of 2005.

Ms Garten said the bird flu strains being detected in Europe were generally clade two strains.

-Reuters

http://www.abc.net.au/news/newsitems/200603/s1596739.htm

Thin Line Stops Bird Flu Spread​

Bird Flu Can't Infect Human Nose or Throat -- Yet
By Daniel DeNoon
WebMD Medical News

March 22, 2006 - A thin red line now keeps people infected with bird flu from infecting others.

This shallow line of defense is a matter of anatomy -- at the cellular level. A new study shows that bird flu can infect cells that line the human lung. But it can't infect most of the cells lining the nose, throat, and sinuses.

Getting the bird flu virus deep in the lung is hard to do, but it can be lethal when it happens. However, spreading the virus is another story. If the virus can't replicate in the nose and throat, it can't be spread by sneezing and coughing.

This may explain why the sparks that could set off a bird flu pandemic -- to date, 184 human infections with the deadly H5N1 strain of bird flu -- have never turned into a conflagration, says study leader Yoshihiro Kawaoka, DVM, PhD. Kawaoka is professor of virology at the University of Wisconsin, Madison.

"It has been an enigma why people get sick and die from H5N1 avian flu virus, but the virus does not spread well in humans," he tells WebMD. "Our finding explains it."

Kawaoka's research team reports the findings in the March 23 issue of the journal Nature.


How Flu Virus Hooks Up

What stops the bird flu? Viruses infect cells by latching on to receptor molecules on the cell surface. Flu viruses bind to sialic acid (SA) receptors.

Most H5N1 viruses -- there are now many strains -- need a receptor in the alpha2,3Gal configuration. In humans, only deep lung cells carry that SA configuration. Nose, throat, and sinus cells have SA in the alpha2,6Gal configuration.

If that doesn't sound very different, it isn't. It would only take a few small mutations for the bird flu virus to be able to latch on to human cells, Kawaoka says.

Flu expert David Topham, PhD, of the University of Rochester, N.Y., says this part of the flu virus mutates rapidly.

"It is relatively easy for the bird flu virus to accommodate such a thing," Topham tells WebMD. "And when people get the infection deep in the lung, there would be selective pressure on the virus to acquire this mutation. So this adaptation to humans might not have to happen in another species. It might occur in humans."

In fact, it's already happened. Kawaoka finds that an H5N1 virus isolated from an infected patient in Hong Kong already was able to hook up to SA receptors throughout the human respiratory system. The world may have dodged a bullet when that virus failed to spread.

Kawaoka says bird flu viruses have to collect other mutations, in addition to the SA-receptor mutation, in order to cause a human pandemic.

"One mutation that makes the avian virus grow better in human cells was found in recent human H5N1 viruses -- which was scary," he says. "We don't know how many mutations in different genes are required for the avian influenza virus to become a human pandemic. That is the problem. We don't know where we are."

http://www.webmd.com/content/article/120/113701?src=RSS_PUBLIC

IMHO, it appears that Niman has identified the final mutation...

S227N H5N1 In Human Upper and Lower Respiratory Tract​

Recombinomics Commentary
March 22, 2006

It is interesting that the A/Hong Kong/213/03 (H5N1) virus, which was isolated from a human and recognizes both SA2,3Gal and SA2,6Gal (ref. 6), bound extensively to both bronchial and alveolar cells.

The above comments in tomorrow’s Nature may explain the large cluster in Turkey associated with S227N detected in the index case. S227N had previously been shown to have reduced affinity for avian receptors (SA2,3Gal) and increased affinity for human receptors (SA2.6Gal). Although the recent paper in Science failed to find increased affinity for SA2,6Gal, the S227N was on a genetic background from an isolate in Vietnam which was distinct from the Hong Kong isolate listed above. In Turkey, the S227N was on a Qinghai strain background, which is different than both the Vietnam or Hong Kong strains used in the lab experiments.

However, the size of the cluster in Turkey suggests that H5N1 was more efficiently transmitted human-to-human, which would be consistent with increased affinity for SA2.6Gal receptors. The H5N1 from the index case was said to be a mixture of S227N and wild type S227. Only wild type was detected in the sister of the index case, in contrast to the Hong Kong isolates which had S227N from both the father and son. However, the Hong Kong isolates were grown on a canine cell line, MDCK, as was the H5N1 used in the Nature paper. It is unclear what was used to isolate H5N1 from the index case and his sister. Isolation of H5N1 on chicken eggs can reduce or eliminate S227N.

However, changes in the levels of S227N and wild type could have been modulated in the patients. H5N1 was isolated from lung tissue, which may have selected against S227N in vivo, since the above data suggests the wild type would grow more efficiently in lung tissue.

Thus, the S227N could have facilitated growth in the upper respiratory tract, while S227 could have been more involved in growth in the lower respiratory tract. Genes encoding S227 could have entered the upper respiratory tract in a pseudo-typed H5N1.

These data could also explain the cluster of cases in Azerbaijan which were fatal, yet efficient transmitted between the index case, her two cousins, and a close friend.

http://www.recombinomics.com/News/03220602/H5N1_S227N_Upper_Lower.html

Random Mutation Explanation of Flu Genetics Is Fatally Flawed​

http://www.recombinomics.com/News/03300602/Random_Mutation_Flawed.html

Recombinomics Commentary
March 30, 2006

The “random mutation” explanation of pandemic or seasonal flu evolution is almost dead. The recent Canadian swine sequences leave little doubt that almost all rapid genetic change in influenza is driven by recombination.

Earlier comments described recombination in PB2 and PA genes of the swine isolates. However, the recombination is in all eight gene segments and in all seven swine isolates. Isolates from the mid-nineties or earlier with exact matches in the recent swine isolates include A/Fukushima/114/96(H3N2), A/Swine/Tennessee/24/77(H1N1), A/Swine/Tennessee/26/77(H1N1), A/Swine/St-Hyacinthe/106/91(H1N1), A/WI/4754/94(H1N1), A/WI/4755/94(H1N1), A/Swine/Wisconsin/3523/88(H1N1), A/Swine/Iowa/930/01(H1N2).

These data show that recombination is very common and the size of the earlier regions get smaller because of further recombinations within recombinants. The data leaves little room for random mutations.

Similarly, recent isolates of the Qinghai H5N1 bird flu strains show single nucleotide polymorphisms overlaid on the Qinghai background. Virtually all of these polymorphisms are well represented in the sequence database and can be found in other H5N1 on migratory bird pathways.

The data indicate the current explanations of influenza evolution are fatally flawed, yet they are the basis of repeated WHO press releases on the looming pandemic.

A full review of the misconceptions driving vaccine development is long overdue.

Link to April Lab report: http://www.timebomb2000.com/vb/showthread.php?t=192010