From Two Mutations, an Important Clue About the Spanish Flu

MHSC · Feb 1, 2007

Pandemic flu may be only two mutations away

19:00 01 February 2007
NewScientist.com news service

Debora MacKenzie

The difference between a flu virus that kills millions, and one that kills only a few comes down to just two amino acid changes, researchers say.

The finding could allow scientists to stay one step ahead of an H5N1 flu pandemic by screening for the specific mutations that would enable it to spread.

A new study investigating the difference between the 1918 pandemic flu virus – which killed at least 50 million people – and a virus which kills but does not spread turned out to be two small mutations on the virus’s surface. Just two amino acids – the building blocks of protein – need to change on the virus’s surface in order to allow it to spread easily between people, the researchers found.

The discovery comes as H5N1 continues to kill. Indonesia this week declared a state of emergency, as it counted its 63rd death. Sub-Saharan Africa confirmed its first death, a 22 year old woman in Lagos, Nigeria.

Nose and throat

Haemagglutinin, the main surface protein on flu viruses, binds to sugars on cells in the nose and lungs; the virus then enters the cells and replicates. Bird flu prefers a sugar called 2,3-sialic acid. Flu adapted to mammals attaches better to 2,6-sialic acid. Mammals have the 2,3 sugar deep in their lungs, but 2,6 in the nose and throat.

H5N1 prefers 2,3. It had been thought that that was why it causes a devastating deep-lung infection in humans, but does not spread between people, because it does not bind and replicate in the nose.

Terrence Tumpey and colleagues at the US Centers of Disease Control in Atlanta, Georgia, US, found this was not how the 1918 virus worked. They reconstructed the virus, and changed one or two amino acids in its haemagglutinin. The combination of both of these changes transform the protein into the one found on the bird flu version of the 1918 virus.
They then gave the viruses to ferrets – animals that get flu in a way most similar to humans. The 1918 virus killed, and spread to other ferrets. One amino acid change, and it still killed, but spread poorly. Two changes, and it killed, but did not spread. Yet all the viruses replicated abundantly in the ferrets’ noses, showing that replication alone does not make a virus transmissible.

Sneeze alert

The unchanged 1918 virus binds to 2,6-sialic acid, while the ones altered by the researchers bound to 2,3. Apparently a virus that binds 2,3-sialic can nevertheless multiply in mammals’ noses after all, a finding echoed last month by scientists in Hong Kong, who found H5N1 replicates well in human nasal cells with no 2,3 sugars.

What a virus needs to spread, the CDC team concluded, is an ability to bind 2,6 sugars, whether or not it needs this to replicate. What this binding does do is not clear. One clue, they speculate, is that ferrets with non-contagious viruses – H5N1, or mutant 1918 – do not sneeze. Contagious ferrets do.

“The cells with 2,3-sialic acid receptors have been associated with the bronchial mucins,” Tumpey told New Scientist. This viscous secretion might inhibit these viruses, and prevent the irritation that causes sneezing, which “may contribute to the spread of influenza, at least in ferrets”.

But what is important is what this tells us about how the next pandemic might begin. The same mutations that made the 1918 flu contagious will not apply to H5N1, as it has a different haemagglutinin. However, the CDC results suggest finding out what mutations make H5N1 bind to 2,6-sialic, as those could make it contagious. We could then watch for those mutations to spot an emerging pandemic early.

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Weblinks

aardvark · Feb 1, 2007

Re: Pandemic flu may be only two mutations away

Re: Pandemic flu may be only two mutations away

This thread captures the same news: http://www.flutrackers.com/forum/showthread.php?t=15842

Here is the corresponding CDC press release:
http://www.cdc.gov/od/oc/media/pressrel/2007/r070201.htm

It looks as if this research is slated for publication in the upcoming issue of Science. The big publications like Science and Nature make advance copies of upcoming issues available to select media outlets, which occassionaly results in cases like this where the media is reporting on something that has not yet been officially published. The full paper should appear on the Science site within a couple of days. Sometimes if they detect a lot of public interest, they'll put an article up for advance web release, but they have not done so yet with this article. So unless we have any select members of the press among us, we'll just have to be wait a few more days to read the details. :tiphat:

Mellie · Feb 1, 2007

Re: Pandemic flu may be only two mutations away

Re: Pandemic flu may be only two mutations away

Here's the New Science article link:

http://www.newscientist.com/article/dn11086-pandemic-flu-may-be-only-two-mutations-away.html

aardvark · Feb 1, 2007

Re: Pandemic flu may be only two mutations away

Re: Pandemic flu may be only two mutations away

turns out we didn't need to wait too long -- Science now has the paper up on their website:

http://www.sciencemag.org/cgi/content/short/315/5812/655

Science 2 February 2007:
Vol. 315. no. 5812, pp. 655 - 659
DOI: 10.1126/science.1136212

Reports

A Two-Amino Acid Change in the Hemagglutinin of the 1918 Influenza Virus Abolishes Transmission

Terrence M. Tumpey,1* Taronna R. Maines,1 Neal Van Hoeven,1 Laurel Glaser,2 Alicia Sol?rzano,2 Claudia Pappas,1,2 Nancy J. Cox,1 David E. Swayne,3 Peter Palese,2 Jacqueline M. Katz,1 Adolfo Garc?a-Sastre2

The 1918 influenza pandemic was a catastrophic series of virus outbreaks that spread across the globe. Here, we show that only a modest change in the 1918 influenza hemagglutinin receptor binding site alters the transmissibility of this pandemic virus. Two amino acid mutations that cause a switch in receptor binding preference from the human -2,6 to the avian -2,3 sialic acid resulted in a virus incapable of respiratory droplet transmission between ferrets but that maintained its lethality and replication efficiency in the upper respiratory tract. Furthermore, poor transmission of a 1918 virus with dual -2,6 and -2,3 specificity suggests that a predominant human -2,6 sialic acid binding preference is essential for optimal transmission of this pandemic virus. These findings confirm an essential role of hemagglutinin receptor specificity for the transmission of influenza viruses among mammals.

1 Influenza Branch, Mailstop G-16, Division of Viral and Ricksettial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA 30333, USA.
2 Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA.
3 Southeast Poultry Research Laboratory, Agricultural Research Laboratory, U.S. Department of Agriculture, 934 College Station Road, Athens, GA 30606, USA.

* To whom correspondence should be addressed. E-mail: tft9@cdc.gov

HenryN · Feb 1, 2007

From Two Mutations, an Important Clue About the Spanish Flu

<TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD align=left>Science 2 February 2007:
Vol. 315. no. 5812, p. 582
DOI: 10.1126/science.315.5812.582

</TD><TD align=right>Prev | Table of Contents | Next

</TD></TR></TBODY></TABLE>
News of the Week

<VARDEF id=TEXT>[SIZE=-1]VIROLOGY:[/SIZE]
From Two Mutations, an Important Clue About the Spanish Flu

Martin Enserink
HIV is lethal but not all that infectious; the common cold spreads easily but is fairly innocuous. The Spanish flu virus of 1918-1919 had the worst qualities of both, which is why it killed more people than World War I did. But although virologists have learned a lot about the combination of genes that made the virus so deadly, they could only speculate why it spread so easily.
No longer. A study published by Science this week (p. 655) confirms what many had suspected: A small change in the virus's hemagglutinin (HA)--a glycoprotein sitting on its surface by the hundreds--makes the 1918 virus more "avian" and unable to transmit between ferrets, even though it still sickened them. Those same changes in reverse may be what started the 1918 catastrophe--and what could kick off the next one as well.
"This is world news," says flu virologist Ron Fouchier of Erasmus Medical Center in Rotterdam, the Netherlands. "This answers the million-dollar question of how an avian virus can become transmissible between mammals." Still, exactly how the change in HA--which required just two point mutations--renders the virus impotent remains unclear, Fouchier says. Nor does it answer an even more urgent question: Could a similar set of mutations turn the bird flu virus H5N1, now devastating poultry in many countries, from an avian scourge into a human nightmare?
The HA in human flu viruses, such as the annual strains now sickening millions in the Northern Hemisphere, preferentially binds to a receptor on host cells that features a sialic acid bound to galactose through a linkage called

-2,6. This receptor predominates in both human and ferret airways. By contrast, avian viruses such as H5N1 have an HA with a slightly different shape that prefers to bind to a sialic acid linked to galactose through an

-2,3 link; these are in the majority in bird guts.
Based on that knowledge, researchers had suggested that the 1918 virus arose when an avian virus acquired mutations that gave it its predilection for

-2,6, thus becoming more "human" in nature. If so, reversing those mutations should be able to "avianize" the 1918 virus and make it unable to transmit among humans, says Terence Tumpey of the U.S. Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, the main author of the new study.

So Tumpey, with colleagues at CDC and Mount Sinai School of Medicine in New York City, took the 1918 virus--which was resurrected over the past decade and is now the subject of intense study (Science, 7 October 2005, p. 28)--and made a few point mutations. One gave it an affinity for both the

-2,3 and

-2,6 receptors. One more switched its preference completely toward

-2,3.

<TABLE cellSpacing=0 cellPadding=0 border=0><TBODY><TR><TD vAlign=top>http://www.sciencemag.org.proxy.library.vcu.edu/cgi/content/full/315/5812/582/F1

Small change. Two point mutations may have been enough to turn an avian virus into the 1918 flu, which killed more people than World War I. [SIZE=-2]CREDIT: CORBIS[/SIZE]

</TD></TR></TBODY></TABLE>

When the researchers inoculated ferrets--the best animal model for human flu--intranasally with high doses of these two viruses, as well as the original 1918 strain, all three caused severe disease. But the ferrets to watch were those living in the cages next to the sick ones. With the original 1918 strain, they, too, became infected and got sick. With the strain that had a mutation that made it bind to both

-2,3 and

-2,6 receptors, transmission was inefficient; two out of three ferrets in adjoining cages developed antibodies, although neither became really ill. In the strain that bound to

-2,3 only, there was no transmission whatsoever.
The study provides the first direct evidence that receptor preference is key to transmission, says virologist Mikhail Matrosovich of the National Institute for Medical Research in London. But why a few point mutations can have such a dramatic effect is less clear, he says. Although

-2,6 receptors predominate in ferrets, they also have

-2,3 receptors, as do humans; that's why the avianized virus was able to infect them. So why couldn't this strain make the jump to the next cage?
One clue lies in studies last year that showed that human cells with

-2,3 receptors occur primarily deep in the lungs, from where the virus may not so easily escape.

-2,6 receptors, in contrast, were found primarily in the upper respiratory tract. Another hint is that the ferrets infected with the avianized virus didn't sneeze, Tumpey says; it's not hard to see why that would reduce transmission in ferrets. Several groups, meanwhile, are trying to find out if H5N1, too, could become a humanized virus through a few mutations in HA. Mutations in other genes are probably necessary as well, says Yoshihiro Kawaoka of the University of Wisconsin, Madison, and the University of Tokyo, and if humankind is lucky, researchers may discover that the combination of changes needed is unlikely to occur in nature. But in any case, knowing in advance what it takes would give scientists something to be on the lookout for in dead birds and human patients, Fouchier says--and ring the alarm bell if necessary.

HenryN · Feb 1, 2007

Re: From Two Mutations, an Important Clue About the Spanish Flu

<TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD align=left>Science 2 February 2007:
Vol. 315. no. 5812, pp. 655 - 659
DOI: 10.1126/science.1136212

</TD><TD align=right>Prev | Table of Contents | Next

</TD></TR></TBODY></TABLE>
Reports

A Two-Amino Acid Change in the Hemagglutinin of the 1918 Influenza Virus Abolishes Transmission

Terrence M. Tumpey,1* Taronna R. Maines,1 Neal Van Hoeven,1 Laurel Glaser,2 Alicia Solórzano,2 Claudia Pappas,1,2 Nancy J. Cox,1 David E. Swayne,3 Peter Palese,2 Jacqueline M. Katz,1 Adolfo García-Sastre2
The 1918 influenza pandemic was a catastrophic series of virus outbreaks that spread across the globe. Here, we show that only a modest change in the 1918 influenza hemagglutinin receptor binding site alters the transmissibility of this pandemic virus. Two amino acid mutations that cause a switch in receptor binding preference from the human

$alpha.gif$

-2,6 to the avian

$alpha.gif$

-2,3 sialic acid resulted in a virus incapable of respiratory droplet transmission between ferrets but that maintained its lethality and replication efficiency in the upper respiratory tract. Furthermore, poor transmission of a 1918 virus with dual

$alpha.gif$

-2,6 and

$alpha.gif$

-2,3 specificity suggests that a predominant human

$alpha.gif$

-2,6 sialic acid binding preference is essential for optimal transmission of this pandemic virus. These findings confirm an essential role of hemagglutinin receptor specificity for the transmission of influenza viruses among mammals. 
[SIZE=-1]1 Influenza Branch, Mailstop G-16, Division of Viral and Ricksettial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA 30333, USA.[/SIZE]
[SIZE=-1]2 Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA.[/SIZE]
[SIZE=-1]3 Southeast Poultry Research Laboratory, Agricultural Research Laboratory, U.S. Department of Agriculture, 934 College Station Road, Athens, GA 30606, USA. [/SIZE]

* To whom correspondence should be addressed. E-mail: tft9@cdc.gov<SCRIPT type=text/javascript></SCRIPT>
The "Spanish" influenza pandemic virus spread globally and resulted in the deaths of up to 50 million people worldwide (1, 2). The ability of this H1N1 pandemic strain to spread rapidly and cause high rates of illness among humans makes it valuable for studying the molecular properties that confer efficient transmissibility of influenza viruses. An influenza virus bearing all eight gene segments of the 1918 pandemic virus was recently generated in cultured cells, was found to be lethal for chicken embryos and mice, and displayed a high-growth phenotype in human lung cells. Furthermore, the 1918 hemagglutinin (HA) and polymerase genes were shown to be essential for maximal virus replication and optimal virulence (3–5). 
Influenza pandemics seem to occur every 10 to 40 years, but the factors that lead to the emergence of pandemic viruses are complex and poorly understood. However, the establishment of efficient and sustained human-to-human transmission of a virus to which humans have little or no preexisting immunity is a fundamental property of pandemic strains (6, 7). Most threatening is the possibility of another pandemic, similar to that experienced in 1918, caused by a novel influenza subtype virus capable of causing severe respiratory disease and death. The avian influenza H5N1 virus, which has resulted in more than 250 human infections (8), has not acquired human influenza virus genes and lacks the ability to spread efficiently from human to human (9, 10). Reassortment of avian H5N1 virus genes with human H3N2 influenza virus genes was shown to be insufficient for transmission of this avian virus (11), suggesting that additional unknown mutations are required for H5N1 to emerge as a pandemic strain. 
The binding of influenza viruses to their target cells is mediated by the viral HA, which recognizes cell surface glycoconjugates containing terminal sialic acid (SA) residues. Avian influenza viruses preferentially bind SA linked to galactose by an

$alpha.gif$

-2,3 linkage (

$alpha.gif$

2,3 SA), which is found in high concentrations on the epithelial cells of the intestine of waterfowl and shorebirds (12). Conversely, human influenza viruses (H1 to H3 subtypes) more readily bind to receptors that contain terminal

$alpha.gif$

-2,6-linked sialyl-galactosyl (

$alpha.gif$

2,6 SA) moieties that are found on the human respiratory tract epithelium (13, 14). The three influenza pandemic viruses of the last century, occurring in 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2), each possessed an HA with a human

$alpha.gif$

2,6 SA binding preference and are thought to have originated from an avian virus possessing the

$alpha.gif$

2,3 SA binding preference (13–16). It has been postulated that the lack of sustained human-to-human transmission of avian influenza H5N1 viruses is due to their

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2,3 SA receptor binding preference (17–19). Higher proportions of

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2,3 SA receptors in the human lower respiratory tract compared with the upper respiratory tract may explain the severity of H5N1 viral pneumonia in humans resulting from H5N1 viral attachment deep in the lungs (17, 19). 
Amino acids at positions 190 and 225 in the 1918 pandemic influenza virus HA determine its receptor binding specificity (15, 16). In this study, we generated recombinant influenza viruses possessing all eight gene segments of the 1918 influenza virus to examine the role of receptor binding specificity on replication, pathogenicity, and transmissibility of this pandemic strain. We generated two variant A/South Carolina/1/18 (SC18) 1918 viruses in which the HA was altered to change the receptor binding specificity from the parental human

$alpha.gif$

2,6 SA (SC18) receptor preference to an avian

$alpha.gif$

2,3 SA receptor preference (AV18) or a mixed

$alpha.gif$

2,6 and

$alpha.gif$

2,3 SA specificity reflecting the A/New York/1/18 (NY18) virus binding specificity. The NY18 virus was a natural variant sequenced from an archived lung tissue sample prepared during autopsy of a patient who died within 6 days of hospitalization in September 1918 (20). The HA corresponding to NY18 virus was made by introducing a single amino acid substitution [Asp225<IMG alt="->" src="http://www.sciencemag.org.proxy.library.vcu.edu/math/rarr.gif" border=0>Gly225 (D225G)] in the SC18 HA. The AV18 virus, which differs by one amino acid from NY18 virus, was made by introducing an additional amino acid change [Asp190<IMG alt="->" src="http://www.sciencemag.org.proxy.library.vcu.edu/math/rarr.gif" border=0>Glu190 (D190E)] within the NY18 HA. Compared with the SC18 virus, the AV18 variant has two amino acid changes (D190E and D225G) in the HA, which matches the conserved avian consensus sequence in the receptor binding site and which converts it to the classic

$alpha.gif$

2,3 SA receptor preference (15). A/Duck/Alberta/35/76 (Dk/Alb) and A/Texas/36/91 (Tx/91) viruses were included in the study as controls representative of an avian H1N1 virus and a human H1N1 virus, respectively. The 1918 viruses were generated by using the previously described reverse genetics system (21–23), and the identities of virus genes in the rescued viruses were confirmed by reverse transcription polymerase chain reaction and sequence analysis. 
The rescued 1918 viruses containing the parental SC18 HA and the two variant HAs had similarly high infectivity titers in Madin-Darby canine kidney (MDCK) cells (Table 1). The receptor-binding properties of the 1918 viruses were confirmed in HA assays by using enzymatically modified chicken red blood cells (CRBCs) that contain either

$alpha.gif$

2,3 or

$alpha.gif$

2,6 SA, as previously described (15). The AV18 virus and the avian Dk/Alb control virus hemagglutinated the

$alpha.gif$

2,3-resialylated CRBCs only, whereas the SC18 virus hemagglutinated the

$alpha.gif$

2,6-resialylated CRBCs only. The NY18 virus hemagglutinated both

$alpha.gif$

2,3- and

$alpha.gif$

2,6-resialylated CRBCs. 

Table 1. Titer of virus stocks prepared on MDCK cells with trypsin (1 µg/ml, Sigma) and incubated at 37°C with 5% CO2 for 48 hours. Hemagglutination assay of viruses used 0.5%

$alpha.gif$

-2,3-resialylated CRBCs,

$alpha.gif$

-2,6-resialylated CRBCs, or untreated CRBCs. The results shown correspond to four hemagglutination units. Similar results were obtained when viruses were adjusted to 8, 16, or 32 hemagglutination units with untreated CRBCs. <TABLE width="100%" border=1><TBODY><TR><TD><TABLE cellSpacing=10 cellPadding=0 width="100%"><TBODY><TR><TD vAlign=top align=left></TD><TD vAlign=top align=middle colSpan=2>Amino acid position (H3 numbering)

<HR noShade SIZE=1></TD><TD vAlign=top align=middle></TD><TD vAlign=top align=middle colSpan=3>Presence or absence of hemagglutination
<HR noShade SIZE=1></TD></TR><TR><TD vAlign=top align=left></TD><TD vAlign=top align=middle>190 </TD><TD vAlign=top align=middle>225 </TD><TD vAlign=top align=middle>Infectivity titer (pfu/ml) </TD><TD vAlign=top align=middle>

$alpha.gif$

2,6 CRBCs </TD><TD vAlign=top align=middle>

$alpha.gif$

2,3 CRBCs </TD><TD vAlign=top align=middle>Untreated CRBCs </TD></TR><TR><TD colSpan=7><HR></TD></TR><TR><TD vAlign=top align=left>SC18 </TD><TD vAlign=top align=middle>D </TD><TD vAlign=top align=middle>D </TD><TD vAlign=top align=middle>4.8 [FONT=arial,helvetica]x[/FONT] 107 </TD><TD vAlign=top align=middle>+ </TD><TD vAlign=top align=middle>- </TD><TD vAlign=top align=middle>+ </TD></TR><TR><TD vAlign=top align=left>NY18 </TD><TD vAlign=top align=middle>D </TD><TD vAlign=top align=middle>G </TD><TD vAlign=top align=middle>3.3 [FONT=arial,helvetica]x[/FONT] 107 </TD><TD vAlign=top align=middle>+ </TD><TD vAlign=top align=middle>+ </TD><TD vAlign=top align=middle>+ </TD></TR><TR><TD vAlign=top align=left>AV18 </TD><TD vAlign=top align=middle>E </TD><TD vAlign=top align=middle>G </TD><TD vAlign=top align=middle>5.0 [FONT=arial,helvetica]x[/FONT] 107 </TD><TD vAlign=top align=middle>- </TD><TD vAlign=top align=middle>+ </TD><TD vAlign=top align=middle>+ </TD></TR><TR><TD vAlign=top align=left>Dk/Alb </TD><TD vAlign=top align=middle>E </TD><TD vAlign=top align=middle>G </TD><TD vAlign=top align=middle>2.2 [FONT=arial,helvetica]x[/FONT] 107 </TD><TD vAlign=top align=middle>- </TD><TD vAlign=top align=middle>+ </TD><TD vAlign=top align=middle>+ </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>

Pathogenesis and transmissibility of the parental 1918 (SC18) virus were evaluated and compared with those of Tx/91 virus with an

$alpha.gif$

2,6 SA receptor binding preference (16) and with those of the avian Dk/Alb virus possessing an

$alpha.gif$

2,3 SA receptor binding preference (Table 1) (24). Ferrets were housed in adjacent cages that prevented direct and indirect contact between animals but allowed spread of influenza virus through the air (11, 25). They were inoculated intranasally with 106 PFU (plaque forming units). One day after infection, three naïve ferrets housed in transmission cages were placed adjacent to each of the three inoculated ferrets (26). Three additional inoculated ferrets from each virus-infected group were killed on day 3 postinoculation (p.i.) for assessment of pathologic and virologic parameters (26). Ferrets inoculated with the parental SC18 virus shed high titers of infectious virus in nasal washes beginning as early as day 1 p.i. [50% egg infectious dose (EID50/ml) from 106.25 to 107.25], and they sustained titers of

$ge.gif$

104.5 EID50/ml for 9 days p.i. (Fig. 1A, left). SC18 virus caused severe disease in all inoculated ferrets starting 2 days p.i.; symptoms included lethargy, anorexia, rhinorrhea, sneezing, severe weight loss (Table 2 and fig S1), and high fever, and two of the three animals died by day 11 p.i. Ferrets inoculated with H1N1 Tx/91 and Dk/Alb also shed high titers of virus in nasal washes (peak titers had EID50/ml values from 105.5 to 106.8), but they were able to clear the virus from the upper respiratory tract by day 9 p.i. (Fig. 1, B and C) after displaying minimal symptoms (Table 2). 


<CENTER><TABLE cellSpacing=0 cellPadding=0 width="95%"><TBODY><TR bgColor=#e1e1e1><TD><TABLE cellSpacing=2 cellPadding=2><TBODY><TR bgColor=#e1e1e1><TD vAlign=top align=middle bgColor=#ffffff>http://www.sciencemag.org.proxy.library.vcu.edu/cgi/content/full/315/5812/655/FIG1 </TD><TD vAlign=top align=left>Fig. 1. Replication in the upper respiratory tract and transmissibility of H1N1 viruses. Three ferrets were inoculated with 106 PFU of SC18 (A), Tx/91 (B), or Dk/Alb (C) virus and housed in individual cages. Naïve ferrets were placed in cages adjoined to those of the inoculated ferrets, and viral shedding in the upper respiratory tract was assessed on alternating days for inoculated (left) and contact (right) ferrets. Results from individual ferrets are represented. Solid and dotted bars of same shade represent a separate ferret pair housed in adjoined cages. The limit of virus detection was 101.2 EID50/ml. </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>

Table 2. Clinical symptoms, virus replication, seroconversion, and transmissibility among ferrets inoculated with H1N1 viruses and among ferrets exposed to the inoculated animals (contacts). The percentage of mean maximum weight loss is shown. NW, nasal wash. <TABLE width="100%" border=1><TBODY><TR><TD><TABLE cellSpacing=10 cellPadding=0 width="100%"><TBODY><TR><TD vAlign=top align=left></TD><TD vAlign=top align=middle colSpan=4>Inoculated ferrets Number with characteristic/total number

<HR noShade SIZE=1></TD><TD vAlign=top align=middle colSpan=3>Contact ferrets Number with characteristic/total number
<HR noShade SIZE=1></TD><TD vAlign=top align=middle rowSpan=2>Respiratory droplet transmission </TD></TR><TR><TD vAlign=top align=left></TD><TD vAlign=top align=middle>Sneezing (day of onset) </TD><TD vAlign=top align=middle>Weight loss (%) </TD><TD vAlign=top align=middle>Virus detected in NW </TD><TD vAlign=top align=middle>Seroconversion (range of HI antibody titer) </TD><TD vAlign=top align=middle>Weight loss (%) </TD><TD vAlign=top align=middle>Virus detected in NW </TD><TD vAlign=top align=middle>Seroconversion (range of HI antibody titer) </TD></TR><TR><TD colSpan=9><HR></TD></TR><TR><TD vAlign=top align=left>SC18 </TD><TD vAlign=top align=middle>3/3 (2) </TD><TD vAlign=top align=middle>3/3 (11.7) </TD><TD vAlign=top align=middle>3/3 </TD><TD vAlign=top align=middle>1/1 (1280)* </TD><TD vAlign=top align=middle>2/3 (15.4) </TD><TD vAlign=top align=middle>3/3 </TD><TD vAlign=top align=middle>3/3 (80-640) </TD><TD vAlign=top align=middle>Efficient </TD></TR><TR><TD vAlign=top align=left>Tx/91 </TD><TD vAlign=top align=middle>3/3 (2) </TD><TD vAlign=top align=middle>3/3 (6.2) </TD><TD vAlign=top align=middle>3/3 </TD><TD vAlign=top align=middle>3/3 (160-640) </TD><TD vAlign=top align=middle>3/3 (3.5) </TD><TD vAlign=top align=middle>3/3 </TD><TD vAlign=top align=middle>3/3 (160-320) </TD><TD vAlign=top align=middle>Efficient </TD></TR><TR><TD vAlign=top align=left>Dk/Alb </TD><TD vAlign=top align=middle>2/3 (5) </TD><TD vAlign=top align=middle>2/3 (1.2) </TD><TD vAlign=top align=middle>3/3 </TD><TD vAlign=top align=middle>3/3 (80-1280) </TD><TD vAlign=top align=middle>0/3 </TD><TD vAlign=top align=middle>0/3 </TD><TD vAlign=top align=middle>0/3 </TD><TD vAlign=top align=middle>None </TD></TR><TR><TD vAlign=top align=left>AV18 </TD><TD vAlign=top align=middle>0/3 </TD><TD vAlign=top align=middle>3/3 (14.7) </TD><TD vAlign=top align=middle>3/3 </TD><TD vAlign=top align=middle>1/1 (640)* </TD><TD vAlign=top align=middle>0/3 </TD><TD vAlign=top align=middle>0/3 </TD><TD vAlign=top align=middle>0/3 </TD><TD vAlign=top align=middle>None </TD></TR><TR><TD vAlign=top align=left>NY18 </TD><TD vAlign=top align=middle>0/3 </TD><TD vAlign=top align=middle>3/3 (18.9) </TD><TD vAlign=top align=middle>3/3 </TD><TD vAlign=top align=middle>2/2 (320-640)

</TD><TD vAlign=top align=middle>1/3 (1.4) </TD><TD vAlign=top align=middle>1/3 </TD><TD vAlign=top align=middle>2/3 (40-80) </TD><TD vAlign=top align=middle>Inefficient </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
* Only one ferret survived and was tested.

$dagger.gif$

Two ferrets survived and were tested.


The human SC18 and Tx/91 viruses efficiently transmitted to each of the three contact ferrets (Fig. 1, A and B, right). The SC18 virus was detected in the contact ferrets as early as day 1 postcontact (p.c.), whereas the Tx/91 virus required 3 to 5 days to achieve detectable virus titers in nasal washes of the Tx/91 contact ferrets. The Tx/91 contact ferrets exhibited little morbidity, whereas all three SC18 contact ferrets exhibited severe signs of illness and weight loss, and one of three contact animals failed to clear the virus before it succumbed to infection on day 6 p.c. In contrast to the efficient spread of SC18 and Tx/91 viruses, the avian Dk/Alb virus was not transmitted to naïve contact ferrets, because virus was not detected in the nasal washes from the contact ferrets at any time. Furthermore, seroconversion was not detected by hemagglutination inhibition (HI) analysis of postexposure sera (Table 2). Both A/Duck/New York/15024/96 and A/Turkey/South Dakota/7034/86, which are representative avian viruses with an

$alpha.gif$

2,3 SA receptor preference, exhibited efficient replication in the upper respiratory tract, but no transmission was detected between ferrets. 
We introduced one– and two–amino acid substitutions into the 1918 virus HA to produce SC18 variants NY18 and AV18, respectively. A switch in receptor specificity from an

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2,6 SA (human) to an

$alpha.gif$

2,3 SA (avian) binding preference abolished the transmissibility of the pandemic virus (Fig. 2 and Table 2). Although ferrets inoculated with AV18 virus exhibited severe illness (Table 2 and fig S1) and shed high titers of infectious virus in nasal washes (Fig. 2A, left), none of the three AV18 contact ferrets had detectable virus in nasal washes, and postexposure sera collected from contact animals lacked antibodies against AV18. The NY18 virus, with dual

$alpha.gif$

2,6 and

$alpha.gif$

2,3 SA specificity, also resulted in severe illness and death among the inoculated ferrets, but it failed to transmit efficiently, as evidenced by the paucity of clinical symptoms and virus shedding among the contact ferrets (Fig. 2B). Two of the three NY18 contact ferrets seroconverted with relatively low HI titers of 40 and 80 (Table 2). The lack of efficient transmission was not due to the inability of the NY18 virus to replicate to high titers in the upper respiratory tract, including the nasal turbinates (Fig. 2B, left, and fig S2). Interestingly, no sneezing was noted among the AV18- and NY18-inoculated ferrets through a 14-day observation period, a finding consistent with the lack of notable sneezing observed in ferrets infected with H5N1 viruses (11). 


<CENTER><TABLE cellSpacing=0 cellPadding=0 width="95%"><TBODY><TR bgColor=#e1e1e1><TD><TABLE cellSpacing=2 cellPadding=2><TBODY><TR bgColor=#e1e1e1><TD vAlign=top align=middle bgColor=#ffffff>http://www.sciencemag.org.proxy.library.vcu.edu/cgi/content/full/315/5812/655/FIG2 </TD><TD vAlign=top align=left>Fig. 2. Respiratory droplet transmissibility of 1918 viruses with mutated HA proteins. Three ferrets were inoculated with 106 PFU of AV18 (A) or NY18 (B) virus and placed in separate cages. Naïve ferrets were placed in cages adjoined to those of the inoculated ferrets, and viral shedding in the upper respiratory tract was assessed on alternating days for inoculated (left) and contact (right) ferrets. Results from individual ferrets are represented. Solid and dotted bars of same shade represent a separate ferret pair housed in adjoined cages. </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>

Despite the differences in transmissibility of the parental 1918 (SC18) virus and the mutant 1918 viruses, similar damage to multiple lung lobes was observed 3 days after intranasal infection (26) (Fig. 3). Ferret lungs infected with SC18, AV18, and NY18 viruses exhibited necrotizing bronchiolitis and moderate to severe alveolitis with edema (Fig. 3, A to E, I, and J). Viral antigen was common in lung tissues, with localization in the upper to lower portions of the bronchial airways, bronchial and bronchiolar epithelium, and hyperplasic epithelium within alveoli (Fig. 3, F to H). Ferrets inoculated with control Tx/91 and Dk/Alb viruses generally showed a lack of significant lung lesions (Fig. 3, K to M). 


<CENTER><TABLE cellSpacing=0 cellPadding=0 width="95%"><TBODY><TR bgColor=#e1e1e1><TD><TABLE cellSpacing=2 cellPadding=2><TBODY><TR bgColor=#e1e1e1><TD vAlign=top align=middle bgColor=#ffffff>http://www.sciencemag.org.proxy.library.vcu.edu/cgi/content/full/315/5812/655/FIG3 </TD><TD vAlign=top align=left>Fig. 3. Photomicrographs of hematoxylin and eosin [(A) to (E) and (I) to (L)] and immunohistochemically [(F) to (H) and (M)] stained lung sections from influenza virus–infected ferrets sampled on day 3 after inoculation. (A to H) Lung sections infected by SC18 virus. (A) Severe necrotizing bronchiolitis with severe diffuse alveolitis and edema. Scale bar indicates 50 µm. (B) Severe diffuse alveolitis; scale bar, 20 µm. (C) Necrotizing bronchiolitis; scale bar, 30 µm. (D) Necrosis and (F) associated influenza viral antigen in submucosal serous glandular epithelium of a bronchus; scale bar, 50 µm. (E) Margination and adhesion of neutrophils to endothelial cells of a pulmonary arteriole; scale bar, 20 µm. (G) Influenza viral antigen in epithelium of a primary bronchiole; scale bar, 50 µm. (H) Viral antigen commonly in macrophages and alveolar epithelial cells; scale bar, 20 µm. (I) NY18 virus; severe diffuse alveolitis with accompanying necrotizing bronchiolitis; scale bar, 50 µm. (J) AV18 virus; diffuse severe alveolitis and edema with necrotizing bronchiolitis; scale bar, 50 µm. (K) Tx/91 virus; normal alveoli; scale bar, 15 µm. (L) Dk/Alb virus, purulent bronchiolitis (p) with peribronchiolar mixed cell inflammation and associated moderate alveolitis (a); scale bar, 50 µm. (M) Dk/Alb viral antigen in bronchial epithelium; scale bar, 30 µm. </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>

Receptor binding, the initial event in influenza virus infection, was a major determinant of virus transmission efficiency of the H1N1 pandemic virus. This work also evaluates the virulence of the 1918 virus in a ferret model, a model that is believed to be more representative than the mouse model of disease caused by influenza viruses in humans. In contrast to other human influenza virus strains, the 1918 virus demonstrated uniquely high virulence and lethality in ferrets. The mutant 1918 virus possessing

$alpha.gif$

2,3 SA receptor binding (AV18) was equally virulent in ferrets as the parental SC18 strain at the dose administered. Remarkably, the AV18 virus replicated in the upper respiratory tract as efficiently as the parental SC18 virus, but it failed to transmit to contact ferrets. Moreover, a human

$alpha.gif$

2,6 SA binding preference is essential for optimal transmission of this exceptionally virulent virus. The introduction of a single mutation that converts the HA to dual

$alpha.gif$

2,6 and

$alpha.gif$

2,3 SA binding specificity (NY18) reduced the high transmissibility observed with the parental 1918 (SC18) virus. This result is consistent with the previously demonstrated lack of transmissibility of an H5N1 2003 virus that possessed dual

$alpha.gif$

2,6 and

$alpha.gif$

2,3 SA specificity due to a naturally acquired mutation at HA residue 223 (H5 numbering; residue 227 by H3 numbering) (11, 27). 
Our findings raise the possibility that, to become more transmissible, the currently circulating avian influenza H5N1 virus may require a receptor binding change to a predominant

$alpha.gif$

2,6 SA binding preference. Such a modification of H5 HA may result in improved virus binding to human tracheal epithelial cells expressing high amounts of terminal

$alpha.gif$

2,6 SA motifs and, simultaneously, in an improved ability to overcome the inhibitory effects of human bronchial mucins associated with

$alpha.gif$

2,3 SA receptors (28). However, mutations that caused a shift from the avian-type to human-type receptor binding specificity for the H1 subtype do not cause an equivalent shift in specificity for the H5 subtype (24). Likewise, the amino acid changes required to alter the H3 HA from an avian- to human-type receptor binding specificity are different from those required for the H1 HA. Therefore, it is likely that different avian HA subtypes have different structural requirements to confer receptor specificity. Thus, it is currently unknown which additional mutations in the H5 HAwould cause a shift to the human-type specificity, which may be required for H5N1 viruses to transmit efficiently among humans. 

[SIZE=+1]References and Notes[/SIZE]

1. W. H. Frost, Public Health Rep. 35, 584 (1920).
2. F. Burnet, E. Clark, Influenza: A Survey of the Last 50 Years in the Light of Modern Work on the Virus of Epidemic Influenza (MacMillan, Melbourne, 1942).
3. T. M. Tumpey et al., Science 310, 77 (2005).[Abstract/Free Full Text]
4. J. C. Kash et al., Nature 443, 578 (2006). [ISI] [Medline]
5. D. Kobasa et al., Nature 431, 703 (2004). [CrossRef] [ISI] [Medline]
6. K. D. Patterson, G. F. Pyle, Bull. Hist. Med. 65, 4 (1991). [ISI] [Medline]
7. C. Viboud et al., Vaccine 24, 6701 (2006). [CrossRef] [ISI] [Medline]
8. World Health Organization, "Epidemic and pandemic alert and response (EPR): Avian influenza" (available at www.who.int/csr/disease/avian_influenza/en/index.html).
9. C. B. Bridges et al., J. Infect. Dis. 185, 1005 (2002). [CrossRef] [ISI] [Medline]
10. J. M. Katz et al., J. Infect. Dis. 180, 1763 (1999). [CrossRef] [ISI] [Medline]
11. T. R. Maines et al., Proc. Natl. Acad. Sci. U.S.A. 103, 12121 (2006).[Abstract/Free Full Text]
12. T. Ito et al., J. Virol. 72, 7367 (1998).[Abstract/Free Full Text]
13. M. Matrosovich et al., J. Virol. 74, 8502 (2000).[Abstract/Free Full Text]
14. R. J. Connor et al., Virology 205, 17 (1994). [CrossRef] [ISI] [Medline]
15. L. Glaser et al., J. Virol. 79, 11533 (2005).[Abstract/Free Full Text]
16. J. Stevens et al., J. Mol. Biol. 355, 1143 (2006). [CrossRef] [ISI] [Medline]
17. K. Shinya et al., Nature 440, 435 (2006). [CrossRef] [ISI] [Medline]
18. M. Matrosovich, N. Zhou, Y. Kawaoka, R. Webster, J. Virol. 73, 1146 (1999).[Abstract/Free Full Text]
19. D. van Riel et al., Science 312, 399 (2006); published online 22 March 2006 (10.1126/science.1125548).[Abstract/Free Full Text]
20. A. H. Reid, T. G. Fanning, J. V. Hultin, J. K. Taubenberger, Proc. Natl. Acad. Sci. U.S.A. 96, 1651 (1999).[Abstract/Free Full Text]
21. E. Fodor et al., J. Virol. 73, 9679 (1999).[Abstract/Free Full Text]
22. C. F. Basler et al., Proc. Natl. Acad. Sci. U.S.A. 98, 2746 (2001).[Abstract/Free Full Text]
23. The 1918 viruses were handled under biosafety level 3 enhanced (BSL3) containment in accordance with guidelines of the National Institutes of Health (NIH) and the Centers for Disease Control and Prevention (CDC) (available at www.cdc.gov/flu/h2n2bsl3.htm) and in accordance with requirements of the U.S. Department of Agriculture (USDA)–CDC select agent program.
24. J. Stevens et al., Science 312, 404 (2006); published online 15 March 2006 (10.1126/science.1124513).[Abstract/Free Full Text]
25. The use of the term "respiratory droplet transmission" throughout this report refers to transmission in the absence of direct or indirect contact and does not imply an understanding of the droplet size involved in virus spread between ferrets. The ability of each virus to undergo respiratory droplet transmission among ferrets was assessed by measuring virus titers in nasal washes from contact animals every other day for 9 days. HI analysis was also performed on postexposure ferret sera collected 18 days p.c. Although only single experiments are reported, there was little variation in the replication and transmissibility among the three inoculated and the three contact ferrets for each of the seven H1N1 viruses tested in this study.
26. Material and methods are available on Science Online.
27. A. Gambaryan et al., Virology 344, 432 (2006). [CrossRef] [ISI] [Medline]
28. G. Lamblin et al., Glycoconj. J. 18, 661 (2001). [CrossRef] [ISI] [Medline]
29. We thank C. Chesley for critical review of the manuscript and J. Beck for technical assistance. This work was partially supported by NIH grant PO1 AI058113 (to A.G.-S.), the Northeast Biodefense Center (U54 AIO57158), and the Center for Investigating Viral Immunity and Antagonism (CIVIA) (U19 AI62623). Work in the A.G.-S. and P.P. laboratories is partially supported by the W. M. Keck Foundation. P.P. is a Senior Scholar of the Ellison Medical Foundation. This work was partially supported by Agriculture Research Service, USDA, Current Research Information System project number 6612-32000-039-00D.

<HR>[SIZE=+2][SIZE=+1]Supporting Online Material[/SIZE] 

Materials and Methods 
SOM Text 
Figs. S1 and S2 
References 

[/SIZE]<HR>Received for publication 12 October 2006. Accepted for publication 13 December 2006.

aardvark · Feb 1, 2007

Re: From Two Mutations, an Important Clue About the Spanish Flu

This is an important experimental piece of new evidence.

Here's a very quick synopsis for anyone who may not care to wade through the full paper:

An engineered 1918 pandemic virus (human) with alpha-2,6 sialic acid binding affinity was lethal and transmitted efficiently in ferrets (as expected). Changes to two amino acids (D225G and D190E) in the HA protein changed affinity from alpha-2,6 to alpha-2,3 sialic acid receptors (again, nothing too new here, this has been reported previously).

The interesting thing is the experimental part. The viruses with alpha-2,3 SA affinity (in other words, the more purely avian viruses) were still deadly and replicated proficiently in ferrets, but did not spread efficiently to ferrets housed in adjacent pens.

The fascinating thing is that both the a-2,6-SA and a-2,3-SA viruses replicated efficiently in the ferret upper respiratory tract (this was somewhat unexpected).... the a-2,3 version just failed to transmit to other ferrets.

The subtext, of course, is that the currently circulating H5N1 virus (which still preferentially binds alpha-2,3 SA receptors) could potentially achieve efficient transmission in mammals through only this change in HA affinity for alpha-2,6 SA vs. alpha-2,3 SA. That's a big piece of speculation, but one that can be tested experimentally. (And it sounds like they're planning to go down that path).

HenryN · Feb 1, 2007

Re: From Two Mutations, an Important Clue About the Spanish Flu

niman said:
<TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD align=left>Science 2 February 2007:
Vol. 315. no. 5812, p. 582
DOI: 10.1126/science.315.5812.582

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News of the Week

<VARDEF id=TEXT>[SIZE=-1]VIROLOGY:[/SIZE]
From Two Mutations, an Important Clue About the Spanish Flu

Martin Enserink
Another hint is that the ferrets infected with the avianized virus didn't sneeze, Tumpey says; it's not hard to see why that would reduce transmission in ferrets. .

The study really confirms what was already suspected:

Preparing for a Pandemic

(WTNH, Nov. 9, 2006 11:00 PM)

http://www.wtnh.com/Global/story.asp?S=5659951&nav=3YeX

"Right now it's already transmitting human to human with families," Dr. Niman said. "There are currently cases in Indonesia and there have been recent cases in Egypt this month."

"The virus is literally one sneeze away from being efficiently transmitted and once that happens it can literally move around the world in a matter of weeks," Dr. Niman said.

sharon sanders · Feb 1, 2007

Re: From Two Mutations, an Important Clue About the Spanish Flu

"..Our findings raise the possibility that, to become more transmissible, the currently circulating avian influenza H5N1 virus may require a receptor binding change to a predominant
$alpha.gif$
2,6 SA binding preference. Such a modification of H5 HA may result in improved virus binding to human tracheal epithelial cells expressing high amounts of terminal

$alpha.gif$

2,6 SA motifs and, simultaneously, in an improved ability to overcome the inhibitory effects of human bronchial mucins associated with

$alpha.gif$

2,3 SA receptors."

How Hemagglutinin Works.

http://www.flutrackers.com/forum/showthread.php?t=3901

4-ABBA · Feb 1, 2007

Dr. David Nabarro spoke of H5N1 needing Two Mutations --a *Year* Ago

Dr. David Nabarro spoke of H5N1 needing Two Mutations --a *Year* Ago

NEWS GLOBALNEWS
Flu pandemic 'two mutations away'

http://english.aljazeera.net/News/archive/archive?ArchiveId=18538

UPDATED ON:
SUNDAY, FEBRUARY 12, 2006
0:21 MECCA TIME, 21:21 GMT

The bird flu virus is only two mutations away from a form that can spread easily between people, sparking a pandemic in which millions could die.

Dr Nabarro has asked nations to prepare for a pandemic

Dr David Nabarro, who heads the UN drive to contain the virus, told weekly Portuguese newspaper Expresso that "only two mutations are needed for it to become easily transmissible among humans".

"I wake up every morning thinking that today could be the day that I will see a report about a strange case of bird flu among humans," he said in the interview published on Saturday.

Italy, Greece

Dr Nabarro's statement came amid reports that the virulent H5N1 bird flu virus had reached Italy and Greece.

The virus was found in swans in three Italian regions: Puglia and Calabria in southern Italy, and Sicily.

In Greece, the H5N1 strain was found in three swans in the north of the country.

Nigeria

In Nigeria, authorities were investigating whether the bird flu virus had spread to humans after several people were reported ill, the health minister said on Saturday.

"I wake up every morning thinking that today could be the day that I will see a report about a strange case of bird flu among humans"

Dr David Nabarro,
UN bird flu panel chief

Eyitayo Lambo, the health minister, said his officials were investigating "one or two cases of reported illnesses" among humans which could be due to bird flu, though none had been confirmed so far.

The H5N1 bird flu virus has killed tens of millions of birds since 2003, and there have been at least 165 confirmed cases of the strain spreading to humans, causing about 90 deaths, mostly in Asia.

Pandemic fears

The virus has spread from Asia to eastern Europe, and this week Nigeria reported Africa's first known outbreak of the deadly strain of the disease.

Experts have long said that the H5N1 virus could mutate into a form that is easily transmitted by humans and spark a global pandemic, potentially killing millions.

Nabarro said he had told governments around the world to prepare for the arrival of a human-to-human strain of the virus "as if this will happen tomorrow".

In 1918, an influenza pandemic that was believed to have originated in birds killed more than 40 million people around the world.

Subsequent pandemics in 1957 and 1968 had lower death rates but still caused widespread disruption.

From Two Mutations, an Important Clue About the Spanish Flu

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