Genetic Compatibility and Virulence of Reassortants of H5N1 and H3N2

[mixin]

Genetic Compatibility and Virulence of Reassortants Derived from Contemporary Avian H5N1 and Human H3N2 Influenza A Viruses

Li-Mei Chen#, C. Todd Davis#, Hong Zhou, Nancy J. Cox, Ruben O. Donis*
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America

Abstract
The segmented structure of the influenza virus genome plays a pivotal role in its adaptation to new hosts and the emergence of pandemics. Despite concerns about the pandemic threat posed by highly pathogenic avian influenza H5N1 viruses, little is known about the biological properties of H5N1 viruses that may emerge following reassortment with contemporary human influenza viruses. In this study, we used reverse genetics to generate the 63 possible virus reassortants derived from H5N1 and H3N2 viruses, containing the H5N1 surface protein genes, and analyzed their viability, replication efficiency, and mouse virulence. Specific constellations of avian–human viral genes proved deleterious for viral replication in cell culture, possibly due to disruption of molecular interaction networks. In particular, striking phenotypes were noted with heterologous polymerase subunits, as well as NP and M, or NS. However, nearly one-half of the reassortants replicated with high efficiency in vitro, revealing a high degree of compatibility between avian and human virus genes. Thirteen reassortants displayed virulent phenotypes in mice and may pose the greatest threat for mammalian hosts. Interestingly, one of the most pathogenic reassortants contained avian PB1, resembling the 1957 and 1968 pandemic viruses. Our results reveal the broad spectrum of phenotypes associated with H5N1/H3N2 reassortment and a possible role for the avian PB1 in the emergence of pandemic influenza. These observations have important implications for risk assessment of H5N1 reassortant viruses detected in surveillance programs.

Author Summary
The influenza pandemics of 1957 and 1968 were caused by hybrid viruses consisting of a mixture of human and avian influenza genes. The introduction of avian genes resulted in a sudden change of the virus surface antigens, allowing its worldwide spread due to lack of immunity in the population. The highly pathogenic avian influenza H5N1 virus has continued its spread in domestic and wild birds in Asia, Europe, and Africa. Although H5N1 infection in humans is rare and person-to-person transmission is very inefficient, the steady accumulation of human cases has raised concern over the possible reassortment between H5N1 and human seasonal influenza resulting in a virus with new surface antigens and pandemic potential. In this study, we used recombinant DNA technology to generate a systematic collection of hybrid viruses (with genes from human and avian viruses) bearing H5N1 surface antigens and analyzed their properties in cell culture and in mice. The H5N1 hybrid viruses revealed a broad range of viability and multiplication capacity in cell cultures. In addition, several H5N1 hybrid viruses were highly virulent in mice. Results from this systematic analysis provide important insight to support risk assessment of reassortant H5N1 avian influenza viruses.

Citation: Chen L-M, Davis CT, Zhou H, Cox NJ, Donis RO (2008) Genetic Compatibility and Virulence of Reassortants Derived from Contemporary Avian H5N1 and Human H3N2 Influenza A Viruses. PLoS Pathog 4(5): e1000072. doi:10.1371/journal.ppat.1000072
Editor: Adolfo Garcia-Sastre, Mount Sinai School of Medicine, United States of America
Received: February 21, 2008; Accepted: April 15, 2008; Published: May 23, 2008
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

Full study here:
http://www.plospathogens.org/article...D8A83268E05350

[gsgs]

1-line summary: "we report a strikingly high level of compatibility between avian and human virus genes"
----------------------------------------------------------------------------------------------------


I haven't yet read the whole paper, but

isn't this contrary to the CDC-reassortment paper from 2006,

where we were told that reassortants are not viable,
often quoted in flu-forums since then ?

I couldn't find any mention of that paper (yet)

-------edit-- I found it now, it's reference 11
http://www.pnas.org/cgi/content/full/103/32/12121

[HenryN]

Originally Posted by gsgs

I haven't yet read the whole paper, but

isn't this contrary to the CDC-reassortment paper from 2006,

where we were told that reassortants are not viable,
often quoted in flu-forums since then ?

I couldn't find any mention of that paper (yet)

Please. These are the same authors.
I think you have misunderstod prior posts on "viability". More reassortants in earlier study (which was smaller) did not replicate as well as the parental strains, and didn't transmit at well as the parental strains.
Once again you are mixing up concepts.

[gsgs]

here the table in computer-readable form and sorted by avian-segments:
(they made a picture-file from it :-( )

A/Wyoming/3/2003(H3N2) and A/Thailand/16/2004(H5N1)


segments
12345678
rescue efficiency
+-
plague-size
----------------
aaaaaaaa,76,2,45
aaaaaaah,72,3,45
aaaaaaha,70,2,40
aaaaaahh,72,1,40
aaaaahaa,68,8,27
aaaahaaa,10,5,0
aaaahaah,43,9,15
aaaahaha,10,5,0
aaaahahh,69,2,42
aahaaaaa,64,3,22
aahaaaah,59,7,25
aahaaaha,38,2,17
aahaaahh,48,7,10
aahahaaa,10,5,0
aahahaah,10,5,0
aahahaha,10,5,0
aahahahh,37,2,12
ahaaaaaa,69,2,40
ahaaaaah,73,1,45
ahaaaaha,69,2,40
ahaaaahh,76,2,42
ahaahaaa,20,3,10
ahaahaah,61,9,17
ahaahaha,23,6,17
ahaahahh,69,5,45
ahhaaaaa,61,10,22
ahhaaaah,44,14,15
ahhaaaha,43,13,20
ahhaaahh,43,5,7
ahhahaaa,10,5,0
ahhahahh,31,10,3
haaaaaaa,66,1,12
haaaaaah,71,1,15
haaaaaha,70,3,40
haaaaahh,75,2,35
haaahaaa,10,5,0
haaahaah,45,6,12
haaahaha,28,7,17
haaahahh,68,6,37
hahaaaaa,70,1,30
hahaaaah,73,3,27
hahaaaha,70,3,40
hahaaahh,72,3,37
hahahaaa,10,5,0
hahahaah,39,3,7
hahahaha,53,3,15
hahahahh,67,3,22
hhaaaaaa,46,8,20
hhaaaaah,67,2,17
hhaaaaha,57,1,35
hhaaaahh,63,8,32
hhaahaaa,10,5,0
hhaahaah,44,1,15
hhaahaha,10,5,0
hhaahahh,45,6,35
hhhaaaaa,53,7,12
hhhaaaah,71,1,17
hhhaaaha,66,7,35
hhhaaahh,72,3,35
hhhahaaa,10,5,0
hhhahaah,48,10,10
hhhahaha,26,4,12
hhhahahh,74,1,25
hhhhhhhh,72,2,45
--------------------

correlation(rescue efficiency,plague-size)=0.84



hhhahahh,74,1,25 : so, just the human H3N2 with HA and NA from the Thai-H5N1 replicated well !
(in mice)
now try ferrets ! Quickly !

[gsgs]

what did that study cost ?

wasn't it worth to get this earlier, in 2006 ?

since when did the authors know partial results ?

did they inform WHO, governments ?

[HenryN]

Originally Posted by gsgs

1-line summary: "we report a strikingly high level of compatibility between avian and human virus genes"
----------------------------------------------------------------------------------------------------


I haven't yet read the whole paper, but

isn't this contrary to the CDC-reassortment paper from 2006,

where we were told that reassortants are not viable,
often quoted in flu-forums since then ?

I couldn't find any mention of that paper (yet)

-------edit-- I found it now, it's reference 11

This paper used a human H3N2 and a clade 1 isolate from 2004, which is one of the inherant limitations of such studies. There are not many human clade 1 cases anymore, and H1N1 is the human serotype that has picked up the H5N1 Tamiflu resistance, which is flying around in clade 2.2.

H5N1 is using MANY more combinations than the CDC.

[gsgs]

from the 2006-paper :


http://www.pnas.org/cgi/content/full/103/32/12121

Table 2. In vitro and in vivo characteristics of avian-human
reassortant influenza viruses


Avian or human virus gene[1] PB2,PB1,PA,HA,NP,NA,M,NS
EID50/ml, log10
pfu/ml
Weight loss (%) [2] No. of inoculated ferrets/total number
Sneezing (day of onset) No. of inoculated ferrets/total number
Virus detected in nasal wash (days p.i.) [3] No.of inoc.ferrets/total
Peak nasal wash titer (day) [4]
Weight loss (%) [6] No.of contact ferrets
Sneezing virus detected in nasal wash No.of contact ferrets
Sero-conversion(HI titer) [7]

hhhhhhhh,9.3,5e6,2/3( 1.4),3/3(2),3/3(1-7) ,5.1(1)
aaaaaaaa,7.5,5e5,3/3(21.1),0/3   ,3/3(1-7+),6.2(5)
hhhahahh,7.8,1e3,1/3( 2.2),0/3   ,3/3(1-7+),3.7(3), 1/3(0.8),0/3,0/3,0/3
aaahahaa,7.8,4e6,3/3( 4.1),3/3(2),3/3(1-5) ,5.2(3)  1/3(0.4),0/3,0/3,1/3(40)
hhhahaaa,7.5,1e7,2/3( 1.7),0/3   ,3/3(1-5) ,4.7(3)  3/3(2.3),0/3,0/3,0/3
hhhahaaa,8.3,4e7,3/3( 0.3),2/3(7),3/3(1-5) ,5.1(3)  0/3     ,0/3,0/3,0/3

[1] Avian genes (A) are from HK486 (H5N1), and human genes (H) are
from Vic75 (H3N2) virus.
[2] The percentage mean maximum weight loss is shown.
[3] +, the endpoint of virus detection in nasal washes was not
determined for all ferrets.
[4] Peak nasal wash titers are expressed as the mean log10 EID50/ml.
[5] rg486:VicRNP was passaged five times in ferrets and then grown in eggs
[6] The percentage mean maximum weight loss is shown
[7] HI assays were done by using Vic75 or HK486 and RBCs from
turkey or horse, respectively


Vic75+486 vs Wyo+THA :

hhhhhhhh,9.3,5e6,  7.2,2,45
aaaaaaaa,7.5,5e5,  7.6,2,45
hhhahahh,7.8,1e3,  7.4,1,25
aaahahaa,7.8,4e6,
hhhahaaa,7.5,1e7,  0
hhhahaaa,8.3,4e7,  0

[HenryN]

Originally Posted by gsgs

here the table in computer-readable form and sorted by avian-segments:
(they made a picture-file from it :-( )

A/Wyoming/3/2003(H3N2) and A/Thailand/16/2004(H5N1)


segments
12345678
----------------
aaaaaaaa,76,2,45
aaaaaaah,72,3,45
aaaaaaha,70,2,40
aaaaaahh,72,1,40
aaaaahaa,68,8,27
aaaahaaa,10,5,0
aaaahaah,43,9,15
aaaahaha,10,5,0
aaaahahh,69,2,42
aahaaaaa,64,3,22
aahaaaah,59,7,25
aahaaaha,38,2,17
aahaaahh,48,7,10
aahahaaa,10,5,0
aahahaah,10,5,0
aahahaha,10,5,0
aahahahh,37,2,12
ahaaaaaa,69,2,40
ahaaaaah,73,1,45
ahaaaaha,69,2,40
ahaaaahh,76,2,42
ahaahaaa,20,3,10
ahaahaah,61,9,17
ahaahaha,23,6,17
ahaahahh,69,5,45
ahhaaaaa,61,10,22
ahhaaaah,44,14,15
ahhaaaha,43,13,20
ahhaaahh,43,5,7
ahhahaaa,10,5,0
ahhahahh,31,10,3
haaaaaaa,66,1,12
haaaaaah,71,1,15
haaaaaha,70,3,40
haaaaahh,75,2,35
haaahaaa,10,5,0
haaahaah,45,6,12
haaahaha,28,7,17
haaahahh,68,6,37
hahaaaaa,70,1,30
hahaaaah,73,3,27
hahaaaha,70,3,40
hahaaahh,72,3,37
hahahaaa,10,5,0
hahahaah,39,3,7
hahahaha,53,3,15
hahahahh,67,3,22
hhaaaaaa,46,8,20
hhaaaaah,67,2,17
hhaaaaha,57,1,35
hhaaaahh,63,8,32
hhaahaaa,10,5,0
hhaahaah,44,1,15
hhaahaha,10,5,0
hhaahahh,45,6,35
hhhaaaaa,53,7,12
hhhaaaah,71,1,17
hhhaaaha,66,7,35
hhhaaahh,72,3,35
hhhahaaa,10,5,0
hhhahaah,48,10,10
hhhahaha,26,4,12
hhhahahh,74,1,25
hhhhhhhh,72,2,45



hhhahahh,74,1,25 : so, just the human H3N2 with HA and NA from the Thai-H5N1 replicated well !
(in mice)
now try ferrets ! Quickly !

Please. You are again posting nonsense. The are MANY pure H5N1's the replicate well in mice, ferrets, AND humans. The issue with H5N1 is TRANSMISSION, not replication.

[HenryN]

Originally Posted by gsgs

what did that study cost ?

wasn't it worth to get this earlier, in 2006 ?

since when did the authors know partial results ?

did they inform WHO, governments ?

Please. The data is PUBLISHED, and it says VERY little. H5N1 is a transmission issue, not a replication issue.

[gsgs]

no replication, no transmission.

Floobiehood had believed, hybrids were probably not viable since
that July-2006 paper. You may remember the discussions
and press-releases.

See also the comments of the authors, who were astonished
themselves.

[HenryN]

Originally Posted by gsgs

no replication, no transmission.

Floobiehood had believed, hybrids were probably not viable since
that July-2006 paper. You may remember the discussions
and press-releases.

See also the comments of the authors, who were astonished
themselves.

Please. The earlier study focused on transmission, and none transmitted as well as pure avian. Others did replicate well. The current study simply shows that the VAST majority don't even replicate well. Only 3 combinations caused major weight loss in mice. Qinghai KILLS mice in 3 days.
You are still posting nonsense,

[gsgs]

OK, I read the whole paper now.
Their "important implication for risk assessment"
is presumably just the usual exaggerations of the very paper's importance
usually seen in the introductions of such papers.
As you say, no transmission was examined/considered.
The surprisingly high number of viable reassortants
is not so worrying, since I assume that in coinfections
many combinations are being tried and filtering the
best one from 256 is not so difficult anyway with so many cells
and viruses.

They found apparantly higher virulence as in 2006 with the
hhhahahh combination but they had mice, while in 2006 it were ferrets.

Transmissiability via droplets is difficult to predict,
and to measure. Maybe it's even rather discontinuous in the
genome. So with mutations or reassortments in a hill-climbing
process you only find local maxima, rarely the global one.
And then it depends on weather, host-habits etc.

No significant increase of the panflu-threatlevel

[HenryN]

Originally Posted by gsgs

OK, I read the whole paper now.
Their "important implication for risk assessment"
is presumably just the usual exaggerations of the very paper's importance
usually seen in the introductions of such papers.
As you say, no transmission was examined/considered.
The surprisingly high number of viable reassortants
is not so worrying, since I assume that in coinfections
many combinations are being tried and filtering the
best one from 256 is not so difficult anyway with so many cells
and viruses.

They found apparantly higher virulence as in 2006 with the
hhhahahh combination but they had mice, while in 2006 it were ferrets.

Transmissiability via droplets is difficult to predict,
and to measure. Maybe it's even rather discontinuous in the
genome. So with mutations or reassortments in a hill-climbing
process you only find local maxima, rarely the global one.
And then it depends on weather, host-habits etc.

No significant increase of the panflu-threatlevel

I am on the road and haven't had a chance to go through the paper, but I suspect these are leftovers, most of which grow, but do little more (three legged dog category). Like the earlier studies, isolates with 100% avian H5N1 beat out avian / human reassortants in vritually all categories, including transmission (which is why WHO consultants are not as interested in H5N1. It doesn't fit their model, so the more they look, the more they are wrong).

However, H5N1 couldn't care less about their model, because its nonsense and based on "hopes and dreams", but hazardous to the world's health.

[gsgs]

Figure 3

segments 12345678 from a:TH04 , h:WY03
LD_50 , lethal dose
MID_50 , mouse infective dose
weight loss
MST , mean survival time
lung
spleen
brain
nasal turbinate

---------------------------------
aaaaaaaa,18,15,198,06,71,19,33,55
aaaaahaa,22,14,255,07,67,23,15,43
hahahahh,25,10,172,07,65,00,11,55
hahaaaha,27,15,218,07,69,11,13,52
haaaaaha,28,15,194,09,77,27,16,39
hhhaaaha,34,15,255,06,60,10,15,44
haaaaahh,35,12,255,07,71,19,11,42
hhaaaaha,35,13,224,08,70,14,08,34
haaaaaaa,35,08,195,09,64,22,22,49
ahaaaaha,38,15,223,08,68,07,00,18
hhhahahh,38,18,189,09,70,14,00,53
hahahaha,38,25,174,13,62,08,00,30
hhaaaaaa,38,22,162,07,69,20,22,43
aaaaaaah,38,20,156,08,68,12,00,35
hahaaaaa,38,22,151,08,68,22,15,54
hahaaahh,44,12,237,22,68,19,00,32
hhhaaaah,44,14,229,22,67,18,09,48
hhaaaaah,44,20,189,22,38,00,00,31
haaahahh,44,15,188,22,68,00,11,50
hhaahahh,44,10,169,22,60,07,09,55
ahaaaaah,44,17,144,22,63,25,10,30
ahaahahh,44,16,140,22,46,00,07,45
hhhaaahh,44,17,135,22,32,00,00,16
ahaaaahh,44,08,120,22,39,00,00,07
aaaahahh,44,25,120,22,68,08,00,54
aahaaaaa,44,44,086,22,16,00,00,00
hhaaaahh,44,13,073,22,59,09,00,41
ahaaaaaa,44,35,073,22,49,15,17,32
hahaaaah,44,35,068,22,12,07,00,39
haaaaaah,44,25,063,22,64,09,07,35
aaaaaahh,44,27,057,22,63,00,08,38
aahaaaha,44,10,035,22,42,00,00,30
haaahaah,44,10,029,22,32,08,00,34
hhhaaaaa,44,15,026,22,41,16,10,35
ahaahaah,44,08,002,22,51,12,10,49
aaaaaaha,44,14,000,22,59,00,00,35
aaaahaah,44,12,000,22,46,11,00,46
aahaaahh,44,25,000,22,47,00,00,00
aahaaaah,44,40,000,22,08,00,00,07
ahhaaaaa,44,44,000,22,10,00,00,00
hhhhhhhh,66,60,000,22,00,00,00,00
---------------------------------

correlation coefficients * 100 :

            1   2   3   4   5   6   7   8 
--------------------------------------------
1.LD_50     --  47 -60  78 -59 -58 -61 -48    
2.MID_50    47  -- -42  27 -66 -34 -25 -59    
3.-weight  -60 -42  -- -67  65  41  42  44    
4.MST       78  27 -67  -- -56 -55 -54 -38    
5.lung     -59 -66  65 -56  --  49  48  69    
6.spleen   -58 -34  41 -55  49  --  50  49    
7.brain    -61 -25  42 -54  48  50  --  53    
8.nose     -48 -59  44 -38  69  49  53  --    
--------------------------------------------
            1   2   3   4   5   6   7   8

best correlation between lethal dose_50 and mean survival time

[Shiloh]

Source: http://canadianpress.google.com/arti...HKailnUqgYJz4g

Study shows hybrids of bird flu and human flu viruses fit well, could occur

17 minutes ago

TORONTO — An experiment mating H5N1 avian flu viruses and a strain of human flu in a laboratory produced a surprising number of hybrid viruses that were biologically fit, a new study reveals.

And while none of the offspring viruses was as virulent as the original H5N1, about one in five were lethal to mice at low doses, showing they retained at least a portion of the power of their dangerous parent.

The work suggests that under the right circumstances - and no one is clear what all of those are - the two types of flu viruses could swap genes in a way that might allow the H5N1 virus to acquire the capacity to trigger a pandemic. That process is called reassortment.

"This study is just showing exactly that: There is a risk this virus can successfully reassort with a human virus," said Richard Webby, director of the World Health Organization's collaborating centre for influenza research at St. Jude Hospital in Memphis, Tenn.

"The problem is we don't know at this stage whether there's a benefit to these H5N1 viruses in doing that."

Nor can anyone say why, if the viruses swapped genes so readily in the laboratory, that hasn't seemed to have happened in the parts of the world where H5N1 has been circulating for years.

"This is the million dollar question," says senior author Dr. Ruben Donis, of the U.S. Centers for Disease Control's influenza division.

Reassortment is one of two ways in which a pandemic virus can evolve. The other is for a bird virus to acquire a number of mutations that allow it to more easily infect people and transmit among them.

The latter, called adaptive mutation, is thought to be the way the 1918 Spanish flu virus emerged. The viruses responsible for the milder pandemics of 1957 and 1968 arose through the mixing of human and avian flu virus genes.

This work, done at the CDC, was conducted to study the reassortment potential of H5N1 and H3N2 viruses. H3N2 is one of two human influenza A viruses that cause disease during flu season.

The study was published in PLoS Pathogens, one of the Public Library of Science journals.

Reassortment studies can be done one of two ways. One involves simultaneously infecting cells with the two viruses and seeing what nature produces. The other involves making viruses by piecing together combinations of synthesized human and avian genes.

"It's like Lego," Donis, head of the molecular virology and vaccines branch, says of this approach, which was the one used for this study.

But this is a game of Lego where it's not clear from looking at the pieces which will go together into a structure that will hold. "We really don't understand the rules of engagement for playing the Legos. We don't know what makes these things connect well or not connect well," he admits.

The researchers created 63 viruses representing the various potential combinations of human and avian internal genes, using an H5N1 virus that circulated in Thailand in 2004 and an H3N2 virus recovered in Wyoming in 2003.

All but one of the hybrids carried the hemagglutinin and neuraminidase genes - the H and N in a flu virus's name of H5N1. The remaining one used the neuraminidase from the human virus, creating an H5N2 virus that grew virtually as well as the H5N1 virus and was almost as lethal in mice.

Once the viruses were made they were placed in a medium to see if and how well they grew. Viruses were then harvested to use to infect mice, to test for virulence.

While 13 of the hybrid viruses either didn't grow or barely grew, the other 50 grew to some degree. And 28 replicated nearly as well as the original H5N1. Donis admits he was surprised by how well the avian and human gene combinations performed.

"I was expecting more incompatibility," he says.

By studying the combinations that succeeded and failed, the scientists were able to start to see patterns of which gene combinations are critical for an H5N1 virus to thrive.

When the most viable viruses were tested in mice, none was as nasty as H5N1. "That's the good news," Donis says, alluding to the fact that if reassortment turns H5N1 into a pandemic strain, the resulting virus could be less virulent than the current version.

Since late 2003 there have been 383 confirmed human cases of H5N1 infection and 241, or 63 per cent, of those people have died.

The virus that most closely matched H5N1 for virulence was one with three avian genes, the hemagglutinin and neuraminidase, plus the PB1 gene combined with five genes from the human virus.

Both the viruses from the 1957 and 1968 pandemics carried an avian PB1 gene. The authors suggest that picking up an avian PB1 gene may be a critical step in a potential pandemic virus arising through reassortment.

But just because the viruses mated successfully in a laboratory doesn't mean those viruses could go on to trigger a pandemic. In order to have that potential, a virus would have to be able to transmit from person to person - a skill that has so far eluded H5N1.

"The bottom line is it comes back down to transmission really being the key," Webby says. "But to say that we understand what are the factors involved in transmission is certainly an overstatement."

Earlier work at the CDC on some H5N1-H3N2 reassortant viruses showed they failed to transmit from infected to uninfected ferrets, an animal often used in flu research.

Donis says his team hopes to test its reassortant viruses in ferrets as well, but is still going through the approvals process.

[HenryN]

More 3 legged dog stories.

[HenryN]

Commentary

http://www.recombinomics.com/News/06..._Failures.html

[tropical]

Originally Posted by Shiloh

Source: http://canadianpress.google.com/arti...HKailnUqgYJz4g

Study shows hybrids of bird flu and human flu viruses fit well, could occur
...
TORONTO — An experiment mating H5N1 avian flu viruses and a strain of human flu in a laboratory produced a surprising number of hybrid viruses that were biologically fit, a new study reveals.
...
The work suggests that under the right circumstances - and no one is clear what all of those are - the two types of flu viruses could swap genes in a way that might allow the H5N1 virus to acquire the capacity to trigger a pandemic. That process is called reassortment.
...
Nor can anyone say why, if the viruses swapped genes so readily in the laboratory, that hasn't seemed to have happened in the parts of the world where H5N1 has been circulating for years.
...
This work, done at the CDC, was conducted to study the reassortment potential of H5N1 and H3N2 viruses. H3N2 is one of two human influenza A viruses that cause disease during flu season.
...
The researchers created 63 viruses representing the various potential combinations of human and avian internal genes, using an H5N1 virus that circulated in Thailand in 2004 and an H3N2 virus recovered in Wyoming in 2003.
...
By studying the combinations that succeeded and failed, the scientists were able to start to see patterns of which gene combinations are critical for an H5N1 virus to thrive.
When the most viable viruses were tested in mice, none was as nasty as H5N1. "That's the good news," Donis says, alluding to the fact that if reassortment turns H5N1 into a pandemic strain, the resulting virus could be less virulent than the current version.
...
Both the viruses from the 1957 and 1968 pandemics carried an avian PB1 gene. The authors suggest that picking up an avian PB1 gene may be a critical step in a potential pandemic virus arising through reassortment.
But just because the viruses mated successfully in a laboratory doesn't mean those viruses could go on to trigger a pandemic. In order to have that potential, a virus would have to be able to transmit from person to person - a skill that has so far eluded H5N1.
"The bottom line is it comes back down to transmission really being the key," Webby says. "But to say that we understand what are the factors involved in transmission is certainly an overstatement."
Earlier work at the CDC on some H5N1-H3N2 reassortant viruses showed they failed to transmit from infected to uninfected ferrets, an animal often used in flu research.

All this reasearch based on suceeds and fails experimenting, apart of the numerous animal deaths, looking from the upper text (the marked statements) nobody would be able to prove what strain would go pandemic, nor if it would be less virulent realy.

The main sure thing is there are in the freezers 63 more lethal avian/human (previously non-existant) viruses - now for real.

[gsgs]

it would be useful to test these viruses for transmissiability in some typical
human community.
However we can't do that in most countries for ethical reasons.

So we need some animals, which can simulate human contacts
and have similarsusceptability to the viruses.

Or we make it less virulent while keeping the transmissiability

Or we simulate it on computer with just some laboratory transmission data

But then, we don't even know how much normal flu transmits
by droplets/airosoles/contact.
I don't understand, why it's so difficult to figure this out.
Maybe it's because of the secrecy, because researchers don't
collaborate well, because doctors don't examine and collect data
about from where patients got the virus.

[HenryN]

Originally Posted by tropical

All this reasearch based on suceeds and fails experimenting, apart of the numerous animal deaths, looking from the upper text (the marked statements) nobody would be able to prove what strain would go pandemic, nor if it would be less virulent realy.

The main sure thing is there are in the freezers 63 more lethal avian/human (previously non-existant) viruses - now for real.

Yes, and they are ALL less lethal than H5N1, which is far more widespread than just freezers.

[tropical]

Originally Posted by niman

Yes, and they are ALL less lethal than H5N1, which is far more widespread than just freezers.

I agree that H5N1 are more widespread, dr. Niman.

You would certainly know better than me.
What I want to said is that seems from the cited text, that now we have in the freezer created avian-human hybrid viruses biologicaly fit ("a surprising number of hybrid viruses that were biologically fit"), at the same time where in nature we have not an biologicaly fit human-avian version capable of transmiting itself?
Or it is?

[HenryN]

Originally Posted by tropical

I agree that H5N1 are more widespread, dr. Niman.

You would certainly know better than me.
What I want to said is that seems from the cited text, that now we have in the freezer created avian-human hybrid viruses biologicaly fit ("a surprising number of hybrid viruses that were biologically fit"), at the same time where in nature we have not an biologicaly fit human-avian version capable of transmiting itself?
Or it is?

Biologically fit only means that they can grow. It doesn't mean that they can compete (and the failure to find such reassortants in nature puts them well into the 3 legged dog category).

[AlaskaDenise]

Originally Posted by niman

Commentary

http://www.recombinomics.com/News/06..._Failures.html

Commentary

More Human Avian Influenza Genetic Reassortment Failures
Recombinomics Commentary 15:20
June 2, 2008

And while none of the offspring viruses was as virulent as the original H5N1, about one in five were lethal to mice at low doses, showing they retained at least a portion of the power of their dangerous parent.

The work suggests that under the right circumstances - and no one is clear what all of those are - the two types of flu viruses could swap genes in a way that might allow the H5N1 virus to acquire the capacity to trigger a pandemic. That process is called reassortment.

"This study is just showing exactly that: There is a risk this virus can successfully reassort with a human virus," said Richard Webby, director of the World Health Organization's collaborating centre for influenza research at St. Jude Hospital in Memphis, Tenn.

"The problem is we don't know at this stage whether there's a benefit to these H5N1 viruses in doing that."

Nor can anyone say why, if the viruses swapped genes so readily in the laboratory, that hasn't seemed to have happened in the parts of the world where H5N1 has been circulating for years.

The above comments on the recent paper in PLOS demonstrates the inherent problem with human / avian reassortants involving H5N1. H5N1 has already evolved into an effective killing machine that not only can kill a wide variety of avian species, but can also kill mammals. This has been demonstrated time and again with humans, as well as mammals that eat H5N1 infected birds. Moreover, H5N1 has evolved into multiple sub-clades capable of producing confirmed fatal infections in humans. Clade 1 has produced fatal human infections in southeast Asia (Vietnam, Thailand, Cambodia). Clade 2 has been divided into three major subclades. Clade 2.1 has produced confirmed fatal infections in Indonesia. Clade 2.2 (Qinghai strain) has produced confirmed fatal infections in Turkey, Iraq, Azerbaijan, Egypt, Nigeria, Pakistan. Clade 2.3 (Fujian strain) has produced fatal infections in China, Vietnam, Laos, Cambodia. All of these fatalities have been in the past few years, demonstrating that relatively minor changes in individual genes can extend the lethality for humans to a variety of H5N1 sub-clades currently in circulation.

The property required to move H5N1 into the pandemic category is not grow in mammals or virulence in mammals, it is efficient transmission between mammals. Virtually all of the clusters in the above countries include human to human transmission between family members or close contacts. However, such transmission chains have generally been limited to a few transmission cycles.

For clade 2.2, most of the clusters have been associated with receptor binding domain changes which enhance affinity for human receptors, and in several cases decrease affinity for avian receptors. However, these changes involve alterations at one or two positions. They do not involve reassortment, which swaps one or more entire genes.

Thus, the examples in the recent PLOS paper are primarily the “B list” of reassortants that are viable, but are usually not lethal in mammals. The small list of those that retain lethality have not been shown to increase transmission. Those candidates, which represent the “A list”, were published earlier in a PNAS paper. However, the “A list” also failed to match the growth or transmission efficiencies of H5N1 with eight avian gene segments, representing naturally evolving H5N1.

The failures of reassortants have led some to question whether H5N1 will develop into a pandemic strain. However, it is the successes in increased transmission efficiencies in H5N1 with receptor binding domain changes in human clusters that suggests a pandemic is only one or two nucleotide changes away.

Ironically, it is an H7N2 human / avian reassortant that has produced enhanced transmission in a ferret model, but the removal of the H7N2 sequences from Genbank shortly after the sequences were made public has created confusion on the true genetic composition of A/New York/107/2003, which was isolated from a case infected in 2002 in New York. The deposited sequences have 3 avian genes (HA, NP, NA) and 4 human genes (PB1, PA, MP, NS). The 8th gene sequence, PB2, wasn’t made public. The enhanced receptor domain binding and ferret to ferret transmission was highlighted in a recent PNAS paper. Moreover, if the submiited gene sequences are correct and the patient tested negative for H3N2, then the infection was likely transmitted human to human.

Although the published results have generated a great deal of media attention, the removal of the underlying sequences has not, and the authors of the study have not publicly commented on the sequences, which are not currently marker with a red banner stating that This record was removed at the submitter's request because the source organism cannot be confirmed.

Ironically, the CDC in Atlanta was involved in both studies, as well as a recent paper in Science on the global spread of H3N2. That paper included sequences, including one submitted by the CDC, which confirmed H3N2 HA recombination in patients in Korea in 2002. Both reassortment and recombination involve swapping of genetic information in hosts co-infected with two or more influenza viruses. While the ressortment swaps whole gene segments, recombination involves swapping portions of genes, which may lead to exchanges of single nucleotide polymorphisms, which is the type of change than can alter binding affinities for human or avian receptors.

The ressortment, or swapping of whole genes, is called genetic shift, while the changes in single nucleotide polymorphisms is called drift. One of the basic tenets of influenza genetics holds that the drift is due almost exclusively to de novo copy errors which happen during viral replication. However, the number and patterns of changes support acquisition via recombination.

Regardless of mechanism, sequence analysis demonstrates that stable small changes are far more frequent than stable reassortants, which is why an H5N1 pandemic is much more likely to involve a change in the receptor binding domain that increases affinity for human receptors, than a swapping of whole avian genes fro human genes.


.

[HenryN]

Originally Posted by tropical

I agree that H5N1 are more widespread, dr. Niman.

You would certainly know better than me.
What I want to said is that seems from the cited text, that now we have in the freezer created avian-human hybrid viruses biologicaly fit ("a surprising number of hybrid viruses that were biologically fit"), at the same time where in nature we have not an biologicaly fit human-avian version capable of transmiting itself?
Or it is?

A human / avian combo in H5N1 has never been reported in the field. There is no evidence to support such a combuination that would be more fit than the current H5N1 with 8 avian genes. Replacing avian genes with human genes weakens the virus. Some don't grow, many grow poorly, and a small number cause significant disease in mice.

These lab experiments, which have been ongoing for a decade, have never created a combination that matched the avian H5N1's abilities to produce disease and transmit in mammals.

The experiments were designed to create a virus that was MORE TRANSMISSIBLE. All such experiments to date have been dismal failures, although many different H5N1's have been used with many different human flu gene combinations.

[mixin]

Niman:

The experiments were designed to create a virus that was MORE TRANSMISSIBLE. All such experiments to date have been dismal failures, although many different H5N1's have been used with many different human flu gene combinations.

Are you positively sure they would announce a success?

[HenryN]

Originally Posted by mixin

Niman:

Are you positively sure they would announce a success?

Please, the hundreds of millions in funding is based on a reassortment success.

They would publish in a heart beat.

[gsgs]

they may have signed secrecy agreements, so if the funding
institute doesn't want to publish then the researchers may not
either.
Think military funding

[HenryN]

Originally Posted by gsgs

they may have signed secrecy agreements, so if the funding
institute doesn't want to publish then the researchers may not
either.
Think military funding

Nonsense.

[HenryN]

Originally Posted by gsgs

they may have signed secrecy agreements, so if the funding
institute doesn't want to publish then the researchers may not
either.
Think military funding

I think you should start a separate internet / conspiracy thread, where your comments would be more relevant.

[mixin]

Originally Posted by niman

Please, the hundreds of millions in funding is based on a reassortment success.

They would publish in a heart beat.

Yes, if that is the reason for funding.

We don't even know the number of labs in the U.S., so I'm not convinced anyone can know exactly what is being done in all of them.

I'm not sure it's a *theory* that there are military contracts that will be kept secret.