Forum: Recombination At Vaccine Summit

I was asked to start on topic to discuss the slides presented at the Vaccine Summit in Boston in August.

Novel Vaccines: Bridging Research Development, and Production

The slides are linked to this page

http://www.recombinomics.com/presentations.html

Many of these slides appear to represent data discussed in prior coomentaries at the Recombinomics website.

These slides have examples of recombination of H5N1, H9N2, and H1N1

H5N1 Hong Kong PB2 Recombinant (slide 11)

H5N1 Hong Kong PB1 Recombinant (slide 12)

H5N1 Hong Kong PA Recombinants (slide 13)

H5N1 Hong Kong NP Recombinant (slide 14)

H9N2 Korea NA Recombinants (slide 15)

Canadian Swine PB2 Recombination (slide 23)

Canadian Swine PA Recombination (slide 24)

Canadian Swine PB1 Haplotypes (slide 26)

Slide 23 lists a cartoon of 7 PB2 genes from swine in Canada in 2003 and 2004

http://www.recombinomics.com/phylo/Canadian_Swine_PB2.html

These sequences have regions of the genes that identical sequences with two or more other swine viruses.

Many of the sequences have regions that match the 1977 sample from Tennessee (in gold) combined with the 1998 samples from North Carolina (in Tar Heel blue).

This is a classical example of recombination.

This commentary descibes data in the PB2 slide

http://www.recombinomics.com/News/03180602/H1_Recombination_Swine_PB2_More.html

More Recombination in PB2 Gene of H1 Canadian Swine

Recombinomics Commentary March 18, 2006

This week new H1 swine sequences from Canada were placed on deposit at GenBank in association with the publication, “ Identification of Human H1N2 and Human-Swine Reassortant H1N2 and H1N1 Influenza A Viruses among Pigs in Ontario, Canada (2003 to 2005)” Seven of the isolates, listed below had an avian PB2 gene and as indicated earlier, several were recombinants and had sequences matching portions of three swine isolates from 1977, 1998, and 2002, also listed below:

A/swine/Alberta/56626/03(H1N1)

A/swine/Ontario/11112/04(H1N1)

A/swine/Ontario/23866/04(H1N1)

A/swine/Ontario/48235/04(H1N2)

A/swine/Ontario/53518/03(H1N1)

A/swine/Ontario/55383/04(H1N2)

A/swine/Ontario/57561/03(H1N1)

A/Swine/Korea/CY02/02(H1N2)

A/Swine/Tennessee/24/77 (H1N1)

A/Swine/North_Carolina/35922/98(H3N2)

As noted earlier, Ontario/11112 was an exact match with North Carolina/35922 for positions 756 through at least 1601 (only a partial sequences from North Carolina was available - sequence numbers adjusted to reflect longer sequences).. Ontario/53518 similarly was an exact match with Korea/CY02 from position 568 to the end of the sequence. Ontario/23866 matched Ontario/53518 through position 568 and then matched Tennessee/24 for positions 1008–1326. These data showed that each of the above recent isolates exactly matched an earlier isolate indicating each was a recombinant. The data also provided evidence for absolute fidelity in copying a large gene segment for 6 years and a shorter segment for 26 years. These data raised serious questions that attributed rapid influenza evolution to random mutation……..

RBD, Thank you for starting an independent thread. I assume from your posts that the way you draw your conclusion is by comparing the sequences and finding long stretches that are the same. I had exactly the same approach to start with, but when I searched the literature, I found that other scientists have been using far more stringent methods to verify whether stretches of identical sequences are sufficient to indicate recombination.

One thing before I go any further. There is unfortunately a fair bit of ambiguity in the use of the term ‘recombination’. As I understand it, Niman uses it to indicate cases where portions of one gene segment, rather than the whole gene or gene segment is exchanged. However, there are authors particularly earlier ones who use the term to mean what we would now call reassortment, ie the exchange of a complete gene segment. The following 2 examples are of this kind:

Evolution of human influenza A viruses in nature: recombination contributes to genetic variation of H1N1 strains. J F Young and P Palese PNAS 1979

Recombination of human influenza A viruses in nature. Bean WJ Jr,Cox NJ, Kendal AP, Nature, 1980

I have written on this topic before. This is the link to a summary of a review paper on recombination in negative sense RNA viruses, which IMO is the most complete review of recombination for this particular group of viruses, which includes influenza A.

The original paper can be found here Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses, Chare et al, Oxford UK

Then there are the instances where recombination was suggested as a mechanism, the most famous one being Recombination in the Hemagglutinin Gene of the 1918 “Spanish Flu”, Gibbs et al, Science 2001, which claimed that part of the 1918 HA was from a swine lineage, but this paper was rebutted by Worobey in Questioning the Evidence for Genetic Recombination in the 1918 “Spanish Flu” Virus, Science 2002, but also summarized in the above paper by Chare et al as follows:

““The difficulties in confirming the presence of recombination in some RNA viruses is most notable with respect to human Influenza A virus. Previously, it was proposed that recombination had occurred in a strain isolated from 1918 (South Carolina/1918) with most of the globular domain (HA1) deriving from a swine lineage, while the ancestry of the stalk region (HA2) was human (Gibbs et al., 2001). Although this recombination event was supported in a number of analytical tests, it was demonstrated later that this apparent recombination could be explained better by a substantial difference in substitution rate between HA1 and HA2, which occurred in the human, but not the swine, form of the virus (Worobey et al., 2002). According to our informative sites test, the alignment used by Gibbs et al. (2001) does contain evidence for recombination. Furthermore, the ML incongruence test provided significant evidence (P<0?005) for a recombination event in the South Carolina/1918 strain at the junction of the HA1 and HA2 regions and at a very similar position in isolate Mongolia/88. However, these incongruent phylogenetic trees did not receive strong bootstrap support and Sawyer’s runs test found no evidence for recombination in these data. At present, therefore, the case for RNA recombination in Influenza A virus remains unproven, with extensive rate variation producing a similar phylogenetic signal in this case.”

I also commented on this here last post

Then there are instances where we actually did find recombination in influenza A viruses. 2 papers are mentioned here at 08:11 These are unique examples and deserve our attention. However, they are both non-homologous recombinations involving the HA cleavage site, a very specific type of change which does require more research, especially this can be the mechanism for sudden emergence of a HPAI or even pandemic virus.

So, to summarize, I’m not saying that recombination does not happen. I am saying, however, that reviews of work by other scientists tells me that there are far more complicated issues that require investigative methods which cover areas where false positive results can happen. See the review by Chare et al for their approach.

The other point that I want to make, is that some of these authors, such as Holmes (co-authoring with Chare) and Worobey, have written extensively on recombination in other viruses ie they are experts on recombination, and far from sceptical about the whole thing. But they are telling us that it is not commonly found in certain types of viruses, including influenza A.

The final point is that even assuming the case for existence of recombination as a significant or even common event in flu viruses, I have yet to see the case made for being able to use this to predict either the future evolution of viruses or to make vaccines. At least I’m not convinced.

Of course, as I said, we are all entitled to our opinions. Here I’m only setting out a summary of several reasons why I form the opinion that I have currently. But if new evidence comes about, of course we should review them. I’m not discounting it completely, just keeping it as one hypothesis that I would like to see some clear resolution some time in the future.

In the meantime, I wouldn’t make my plans based on them. But then that’s just me. :-)

Recombination is well defined and easily seen in the slides. There is no ambiguity.

anon 22 you are generous to concede that recombination can, in rare cases, occur. You end by writing “in the meantime, I wouldn’t make my plans based on them. But then that’s just me.”

By banning Niman from the site, you are doing much more than exercising your right to an opinion on the matter. Your are depriving us from being exposed to his. We don’t need your protection. There is plenty on this site that is of dubious merit, and we are given the choice as to whether we wish to plow through it or not. To make a special case of Niman, and to “protect” us from his ideas is both disingenuous and condescending: we can make our own judgements on the merits of his methods, data analysis and conclusions.

On the off chance that Niman is right, and you are wrong, don’t you think that members of the wiki community should be provided access to those ideas, and be offered the chance to query Niman directly?

Please, the stakes are way too high for personal grudges of any kind to impinge on the free flow of information.

ducksoup – at 15:08

You are free to ask that they open a comment section at Recombinomics. Come back and tell us when they do. End of dicussion.

anon_22, The swine slides do not show regions of similarity. They show regions of identity. The data is recombination.

The slide on PA

http://www.recombinomics.com/phylo/Canadian_Swine_PA.html

has long regions of identity than span the gene and include HA1 and HA2.

As noted earlier, there is no ambiguity.

anon_22.

The above comment should have said the gene spanning was the equivalent of HA1 and HA2 (the slide linked was for PA, but the regions of identity covered the who gene and were not due to conserved regions).

You are confusing “similarity” in the example you cited for the 1918 sequences, with the “identity” in the slides linked above.

There is no ambiguity because the matches are exact, and they are with known sequences.

There is no doubt that the examples are homologous recombination ad the examples in the slide cover two genes in seven isolates collected in two province in two years in natural infections.

It really does not get any clearer.

DemfromCt at 15:21

I don’t see why what Niman chooses or doesn’t choose to do on his website has any bearing on his right to present his views here. Note that emerging data from this potentially pandemic virus strain support his view, and are less compatible with the dogma. The reason that all of us come back to this website is because we recognize the stakes. After all this is over, will you be able defend the exclusion of explanatory frameworks because of breaches in etiquette?

Think about it.

ducksoup at 16:52:

The subject of Niman has been discussed endlessly and at this point, I don’t see the issue being revisited. Meanwhile, it’s off topic for this thread. That’s pretty much what Dem meant by “End of discussion.”

pogge

Its not off topic! We’re discussing his data!

Do so.

OTOH, anyone’s banning is the prerogative of the mods and not up for discussion, so keep your focus. Should it stray on to the topic of banning, the thread will be closed.

Is that crystal clear?

RBD and ducksoup,

Thank you for your comments. Let me refer you to the methodology in Chare et al for insights on the discerning the relevance or significance (or not) of similarity or identify in sequences.

ducksoup – at 15:08,

Please, the stakes are way too high for personal grudges of any kind to impinge on the free flow of information.

Thank you for your remarks. In no way are we impinging on anybody’s free access to information. Niman’s ideas are freely available everywhere on the internet, and available here too. This thread is an example.

Yes, the stakes are too high for personal grudges. They are also too high to speak anything but exactly what I mean. There are times when I am sloppy with words. Not so when I say “I wouldn’t make my plans based on them.” But, as I said, that’s just me.

And if I ever change my mind, I will post and let everyone know asap.

Thanks for listening.

anon_22, Sequences in one gene segment exactly matching two distinct sources is the result of homologous recombination.

RBD – at 18:11

Fine. We can agree to disagree.

anon_22, There is a difference between similarity and identity. The commments of Chare et al, were on similarity and rates of change.

The data on the slides represent identities, which is why there is no ambiguity and no need for analysis of rates of change.

The data in the slides are exact matches, which is quite different than similarities analyzed by rates of change, which is the subject of the papers you pasted onto this thread.

Thus, the issue really is on the data on the slides (exact matches), not papers discussing other issues, like rates of change.

Thanks, RBD, for reporting on the Summit. Did they talk about Ilara Capula? There was quite a nice article in the current Science about her efforts to get the witheld sequences released. She seems one fine lady. It seems clear to me that, as sequences are released, they fit into the framework being developed by Niman, and not into the framework developed by Webster. It also seems apparent, judging by their 6-month lags in releasing papers, and that without releasing the sequences, that the Webster crowd do not feel any sense of urgency, because at heart they believe that this is a stochastic, random process that is slow and stupid, and they believe they can stay ahead of it. On the other hand, Niman has a sense of urgency, because if the changes proceed by recombination, then only a very few dual infections are required to complete the process. So far Niman has made at least two important predictions that have been proved by sequence data. Webster can’t make that claim, because random processes can’t be predicted by definition. Well, it oughtn’t be very long before we know, one way or the other.

disgruntled,

I heard the story about the hoarding discussion, which is quite interesting in light of the recent PNAS and Nature papers which set a new personal best on hoarding by WHO consultants.

The issue came up in the panel discussion. The meeting was on vaccines in general, not just pandemic influenza, so one of the questions from the audience was on publication of failed approaches. Some wanted companies to publish failures, so others wouldn’t waste time of money on dead end approaches. They were trade secrete considerations on companies withholding negative data.

Niman brought up the withholding of positive data, like the sequences, which were required for chosing appropriate targets. The NIH reprentative on the panel acknowledge the support for the NIAID flu sequencing project, which generates sequences at no charge, as long as the sequences are made public. He noted that there had been “sharing” issues in the past, but they had been resolved.

That of course was before the PNAS and Nature papers, which are now withholding sequences AFTER publication.

http://www.recombinomics.com/News/11210602/H5N1_Hoarding_Post.html

Now is the time to pressure the hoarders, because is is quite clear that the sequences are important to the understanding of H5N1 evolution and vaccine targets, and the hoaders will hoard as much as possible.

Hey guys, lighten up.

Any pharmaceutical companies buying the Niman theory? Surely there’s lots of money to be made. Don’t like big pharma? No problem. I don’t like them either. Perhaps those who believe this Recombinomics theory can put their money where their mouths are and invest in a venture capital fund to develop Niman Inside Vaccine, just like Intel Inside in your personal computer. No one believed personal computers will be this big an industry.

I think I am on topic. This is about Vaccine Summit, right? It was August, we are now in November. Where is the vaccine that predicts what the pandemic virus looks like? We need it before the pandemic hits us. So many lives will be saved that a Nobel Prize will be waiting. With such a vaccine, who cares about a pandemic - no need to prep any more. Who cares about releasing sequences - it can be predicted. This is bigger than Google. The garage will become a museum. TPTB, are you listening?

Sorry, I don’t have any money to invest. Some one else can get rich. I need the funds for my preps.

For those who don’t want to read through the pasted articles that most will have no clue about what was posted, the issue is really not very complex. The pasted text is regarding recombination in the 1918 pandemic strain.

The original paper indicated part of HA was like one virus, and part like another virus. The response was that different regions of HA evolved at different rates.

The discussion on 1918 really has nothing to do with the slides at the vaccine meeting, because the areas color coded are exact matches with other known viruses.

That is why there really is no controversy. The color coded regions (which are large) are exact matches and therefore represent recombination.

There are few, if any scientists, who understand the color codes (or verfiy the identities for themselves), who would not agree that the data represents homologous recombination.

see here

 for a more extensive list of recombinations

which also shows some lack of less obvious recombinations, which we would expect if recombination were really frequent. I guess, they are almost as frequent as reassortments.

Yes, there are quite a few examples that are “obvious” including those presented at the Vaccine Summit. The daat in the linked slides are the type of data that really only has three viable explanations. It is homologous recombination. The cartoons are misrepresnting the data. The sequencing lab made dozens if not hundreds of errors.

The examples have nothing to do with the 1918 controversy or any of the other pasted papers, some of which were from the early eighties, before there was much sequnece data or before it was known that influenza was a segmented genome.

The amount of flu H5N1 sequence data has grown markedly since 2004, even with the hoarding, so the recombination is becoming increasingly obvious to those who actually look at the data, instead of posting old articles identifed through medline searches.

For recombination to occur, a given cell would have to be infected simultaneously with two distinct and different viruses. Question: Has this been tested in vitro to see what the probability is this will happen, using a variety of different viruses? Cells have lots of “landing sites”, but generally a virus is pretty host-cell specific, and thus the likelihood of two different viruses infecting a single cell would seem to be limited unless they were closely related. Yet H5N1 has been found infecting all sorts of different tissues and organs throughout the body of both birds and mammals, so it may be more likely to be involved in a co-infection/recombination situation than a virus that was highly restricted to one type of cell. Niman’s evidence for recombination, while strong, is gathered from incomplete records derived from genetic material gathered after the fact of infection, and thus would tend to be somewhat circumstantial in nature. I would think that carefully controlled lab research would be warranted to support his theory.

The recnt Nature papaer on receptor binding domain changes had multiple clones from teh same indovidual, and those clones probably only represents a subset of what was in the host. Similarly, H5N3 and H6N2 were isolated from H5N1 positive birds in the United States. Dual infections are very common for infleunza, and dual infections are also required for reassortmant.

Mary in Hawaii – at 23:30

Please save a review of the concept of quasi-species in the last (I think) of the Cytokinic Dysregulation threads in my NS1 Profile link fixed - DemFromCT

As RBD mentioned, recent papers list a number of H5N1 variants in a single patient. The FluTrackers show a nice listing. Scan the top for the word clone.

Our tools are not very sophisticated at the moment in detailing the quasi-species variants; however, I expect to start seeing labs pay much more attention to fuzzy peaks during sequencing.

NS1 makes a Prediction:

In 5 years or less, we will develop tools to easily detect and sequence in one pass the dozens of viable Influenza strains in a single host.

NS1 Profile

Cytokinic Dysregulation 4 Posts starting at 2006–09–22.

NS1 at 23:55 I scanned the abstract you cite regarding viral quasi-species, but didn’t really see the relevance to H5N1 recombination. It was pretty abstruse, but seemed to be making a case for how chronic infection with a virus such as Hepatitis can, through the ongoing action of the mutagenic viral RNA polymerase, affect body cells’ normal membrane receptors leading to autoimmune diseases etc. Like I said, I only scanned and the wording was extremely specialized, so maybe I missed your point.

  However regarding your statement: “As RBD mentioned, recent papers list a number of H5N1 variants in a single patient.” I wanted to ask, are these H5N1 variants the result of recombination among clades of H5N1, or is it evidence of recombination between H5N1 and some other viruses such as Influenza B, swine flu or any of the other H-N flu variations? Also, is the fact of having H5N1 variants in a single patient actually definitive proof of having these two variants inside a single cell of that patient? (which is where they must be for recombination during the viral genetic replication phase to take place.) In any case, I still think my point regarding doing in vitro testing with different clades of H5N1, as well as H5N1 and different viruses, inoculated into tissue cultures of different cell types might give a clearer picture of just how much recombination is potentially taking place in living hosts.

Mary,

You are right on target concerning the need for wider testing protocols with higher individual specificities, especially concerning tissue cultures.

The quasi-species concept is raised because my personal belief is that many infections generate a diverse set of clear individual strains in any one host. I’m merely noting that our surveillance rarely goes beyond the first sequence that we find in a host.

Recombination as an outcome seems clear to me and many others. Recombination as a mechanism, method or cause continues to await the more terse definitions required by many naysayers.

The proof is in the sequences.

Though we cannot observe in a wild infection that donor strain A and donor strain B cross to form Recombinant AB, we can observe the availability of A and B in the geographic region and the outcome of Recombinant AB.

I am not segmenting my studies by clade, only by the H5N1 grouping versus all other seasonal and HPAI. My primary concern is very much along the lines of H5N1 recombining with a seasonal influenza as we’re seeing with influenza B now. Zoonoses run a close second.

Demonstrating multiple strains from a single host does not prove recombination in any way. A careful study of the sequences illuminates that question. A host could be infected initially by multiple strains that reproduce independently. Or a host may be singly infected and have multiple lineages develop due to variable host environment parameters, including gene expression and application of anti-viral drugs or plants. Many options.

Mary in Hawaii, In vitro tests may giev additional information on identity requirements, but the amount of recombination will come from a robust database.

The issues of dual infections and selection pressures determine recombination rates in living hosts in nature.

NS1, RBD, Mary in Hawaii et al,

Your open-minded analysis and discourse on this thread is very, very much appreciated. Please consider an ongoing Recombination thread if the mods feel this thread drifts to far from the original posts. Thanks again.

The mods have never minded discussions of recombination. And given the title, it’d be tough to stray if that’s the topic. ;-)

Homesteader – at 06:51

It isn’t so much open-mindedness, it’s courtesy. ;-) We have written and gotten the CDC to correct and add recombination to possible mechanisms of pandemic evolution in their Q&A on the website. The question has not been (at least here) whether recombination happens. The question is how important a mechanism is it, and can it lead to pandemic? The alternate and more common question (out there) is whether it can predict viral evolution.

Some are convinced, the jury is still out for others. There is absolutely no need to be condsescending, insulting or dismissive of one camp by the other. THAT is true open-mindedness. ;-)

NS1, RBD, Mary in Hawaii et al,

I agree with Homesteader

Your open-minded analysis and discourse on this thread is very, very much appreciated.

I have a question. At the recent ASTHO meeting in Syracuse, several questions were asked regarding potential pandemic vaccines. I will share them here because I think that linking the science to it’s potential practical results might be very interesting to many people.

One question asked concerned why we must always get a new seasonal flu vaccine each year based on the strains conjectured to be circulating during any upcoming year. How would an understanding of recombination change the way we go about formulating the standard seasonal flu vaccine, or a pandemic vaccine? How will it help us do so in advance?

A second question concerned whether a “universal flu vaccine” was possible. Dr. Bruce Gellin took that question, and remarked that a universal flu vaccine was, in fact, the gold standard to which they were working. The new DHHS pandemic plan update #3 also features the potential for a universal vaccine prominently in its pages, which I found surprising. Will an understanding of recombination help in the effort to formulate such a “universal vaccine” (if indeed such a vaccine is possible).

Thanks everybody for the very interesting and congenial discussion - it is a valuable one.

This slide addresses recombination and seasonal flu:

http://www.recombinomics.com/phylo/Canadian_Swine_PB1.html

PB1 is used, because the Canadian swine have a human PB1. The changes in human H3N2 sequences are listed and the number of patients with the listed sequence is displayed by year (numbers in red are human H1N1).

The gold background represents the swine sequences, which match human PB1 in the mid-nineties. The center panel shows the swine (human mid-nineties) sequences disappearing for several years, only to reappear in recent isolates. This type of reappearance of all three changes simultaneously signals recombination.

The is true for the third panel which shows changes in 7 positons chnages between the dominant haplotype for 2003 compared to 2004. This type of dramatic change in a single year also signals recombination involving a non-human reservoir.

All changes are transitions.

In the response to the obvious interest from many people, I’m doing another more extensive review of methodology. But I cannot find Niman’s methodology, not explicitly stated anyhow.

Can anyone give me links, descriptions, etc as to how he arrives at his conclusions? Thanks.

The colors on the table represent the region of the isolate in question that exactly matches the designated sequence. All of the sequences are public and available at Genbank or Los Alamos. Sequences can be aligned with ClustalW. The sequence is the sequence and the exact matches are exact matches. There are no statistics, phylogentic trees, or data manipulation required.

A match means the sequence is EXACTLY the same at EVERY position for the region depicted on the slides.

The matches don’t mean the region is similar, it means the region is EXACTLY the same.

If T=T, it is a match.

If C=C, it is a match.

If A=A, it is a match.

If G=G, it is a match.

Pixie, The recombination does not create a universal vaccine, it is used to create a vaccine against an emerging sequences. The emerging sequences come from the parental strains, so if the parents are known, the new sequence can be predicted.

This was done before H5N1 flew into the Middle East. The H5N1 could acquire S227N by recombining with H9N2, which was endemic in the Middle East. The recombination could be predicted down to the specific nucleotide that would be acquired.

The prediction, made October 22, 2005

http://www.recombinomics.com/News/10220501/H5N1_H9N2_Recombination.html

was confirmed in January, 2006 when the H5N1 from the index case in Turkey was sequenced. That sequence, as well as a second sequence in Turkey and a sequence in Egypt, had S227N, which was formed by the predicted nucleotide change.

Predictions for vaccine targets are based on the prevalent sequences in the region and are dependent on a full and public sequence database, which is why the hoarding of the sequences by WHO is a concern.

http://www.recombinomics.com/News/11220601/H5N1_Hoarding_Azerbaijan.html

Mary in Hawaii – at 23:30

I agree with you, seems that there is strong evidence of recombination in the evolution of this virus. Even a very diligent, reasonably intelligent layperson can see that in the sequence data.

One would hope that scientists willing to work together collaboratively in the greater good would also look at the data and determine for themselves whether there is evidence for recombination.

One would further hope that all of these scientists would put their own petty focus on self-interests and collaborate to do the research to validate this, and, together possibly formulate a solution before the virus decimates our species.

If they do not do this, there may not be anybody around who cares whose theory was right.

The question, of course, is that is this kind of matching of alignment sufficient proof of recombination? The reason why I ask this question, is that over many months of re-visiting this issue, and searching many publications, I have yet found any other scientist using this as the definitive method.

That was the reason why I started to question his methods. That is the reason why I’m still looking for answers.

I’m still looking. I will post when I’m ready. In the meantime, anyone else with further information on Niman’s methodology beyond matching, please post.

For example, this paper A novel approach to detecting and measuring recombination: insights into evolution in viruses, bacteria, and mitochondria, Worobey M gives a detailed description of several methods and issues. The paper is free, as are all 16 papers on recombination on this site from the University of Oxford, Department of Zoology Evolutionary Biology Group.

A novel approach to detecting and measuring recombination: insights into evolution in viruses, bacteria, and mitochondria Worobey M (2001) Mol Biol Evol 18, 1425–1434.

ABSTRACT:

An accurate estimate of the extent of recombination is important whenever phylogenetic methods are applied to potentially recombining nucleotide sequences. Here, data sets from viruses, bacteria, and mitochondria were examined for deviations from clonality using a new approach for detecting and measuring recombination. The apparent rate heterogeneity (ARH) among sites in a sequence alignment can be inflated as an artifact of recombination. However, the composition of polymorphic sites will differ in a data set with recombination-generated ARH versus a clonal data set that exhibits the equivalent degree of rate heterogeneity. This is because recombinant data sets, encompassing regions of conflicting phylogenetic history, tend to yield starlike trees that are superficially similar to those inferred from clonal data sets with weak phylogenetic signal throughout. Specifically, a recombinant data set will be unexpectedly rich in conflicting phylogenetic information compared with clonally generated data sets supporting the same tree shape. Its value of q-defined as the proportion of two-state parsimony-informative sites to all polymorphic sites-will be greater than that expected for nonrecombinant data. The method proposed here, the informative-sites test, compares the value of q against a null distribution of values found using Monte Carlo-simulated data evolved under the null hypothesis of clonality. A significant excess of q indicates that the assumption of clonality is not valid and hence that the ARH in the data is at least partly an artifact of recombination. Investigations of the procedure using simulated sequences indicated that it can successfully detect and measure recombination and that it is unlikely to produce false positives. Simulations also showed that for recombinant data, naive use of maximum-likelihood models incorporating rate heterogeneity can lead to overestimation of the time to the most recent common ancestor. Application of the test to real data revealed for the first time that populations of viruses, like those of bacteria, can be brought close to complete linkage equilibrium by pervasive recombination. On the other hand, the test did not reject the hypothesis of clonality when applied to a data set from the coding region of human mitochondrial DNA, despite its high level of ARH and homoplasy.

There is no heterogeneity. The matches are EXACT.

A=A

T=T

C=C

G=G

Yes, pattern matching is sufficient proof of recombination if the sample size is sufficiently large. A ‘big enough’ segment to match is on the order of 18 base pairs or more. It’s similar to a Google search: three-letter words are useless, there are too many, but certain combinations of larger words, such as ‘sentitious hymn’ only generate a two hits. So to emulate his method, here is all you do:

Assume that nucleotide changes from a ‘child’ are inherited from ‘parent’ strains. Start with a single sequence of interest. Select a segment, then select a contiguous set of nucleotides between 12 and 50 base pairs long. Run it through BLAST and get the matches. If there are way too many matches, the string is not very unique, so discard it and try another. The right sort of string yields several candidate parent sequences. Save that list. Now, go back and select another contiguous string, from another segment. If the first one was from HA, then select another one from NA. Get a list of matches. If there are too few or too many, select other strings until they yield a collection of sequences that is neither too long or too short. Save that list of candidate parents.

Repeat for many short segments, and all gene segments.

Now you have a collection of lists of nearest match parent sequences for independent queries on a single child sequence.

If all the lists contain the same parental sequences, and the same one or two ‘parents’ show up in the lists for a given ‘child’, then look and see how the child matches the parents. Is the child an even mix of the two parents, with 50% of its nucleotides from one parent, and 50% from the other? Or is there one parent with 90% of the parent sequences, and 10% from the other? Or some other combination? Does the timing work out? Does the location work out? For example, were the parent strains both in Qinghai in 2003?

After doing hundreds of BLAST checks, certain patterns start to emerge. Certain strings start to become traceable in space and time. Niman probably has a list of a hundred subsequences that he uses in his searches to trace lineages.

He also searces on minor variants of the strings, and even these minor variants show definite patterns in distribution in space and time.

Perhaps people don’t use this method much because it isn’t glamorous, and isn’t very adaptable to mechanization. It doesn’t uses statistics or big programs. Just methodical, patient rummaging of the database—data mining— and pattern recogniton.

RBD,

You are misunderstanding the ARH, apparent rate of heterogeneity. It is not heterogeneity between one sequence and another sequence, but the rate of heterogeneity along the same sequence. This rate is variable, but more variable in a recombinant than in a clonal set.

I understand what you are saying. What I am asking you to consider is what methods are other scientists using and why are they doing it that way?

anon_22,

The slides are well labeled. The name of the isolate is on the left. The nucleotide position is at the top. The color coding is at the bottom. Looking at the sequence might be easier than multiple medline searches or copying and pasting data that very few who are reading this thread will understand and have no relevance to the slides.

Each slide is a relatively simple gene segment from published influenza sequences. The matches are EXACT. The recombination is OBVIOUS.

The regions color coded are EXACT matches. If you find any errors in the slides, please posts them. The scientists at the Vaccine meeting had no problem seeing that the slides represented homologous recombination. The examples given are in the OBVIOUS category.

Please forgive my scientific ignorance in these matters, and please excercise as much restraint as you can muster before you jump down my throat for getting this wrong (lol) but, as I understand it, sequences are made up of genes. Genes are made up of nucleotides, which can be represented by the letters A T C G. (I hope I’ve got it right so far!)

Genes are made up of hundreds(?) or more of these little nucleotides. Nucleic acid. A,T,C and G. (I’m just clarifying this in my mind, I know that you know this.)

So each region(?) of a gene sequence is assigned a position in the sequence (hence the term sequence! lol)

If you take some gene sequences and run them through a program such as ClustalW (I’ve actually done this, and it is quite interesting and was surprisingly easy) you will get an output that is a colorful representation of these gene sequences with the positions (nucleotides?) all lines up with each other. I must confess, I do not yet know what the color coding signifies. But you can see that in these sequences, they almost all match perfectly, except for a few, and usually far between individual position in the sequence.

Now, how could this happen? Obviously not be reassortment, could it? I thought that entire genes are swapped for this type of evolution of a virus, isn’t that right? Not just individual nucleotides? Again, if anyone could correct me, I would appreciate it, because I am just learning.

So, if it’s not reassortment, what else could it be. Mutation? Do genes mutate that fast? If one understands the quasispecies model, I would think not.

So, what is left? Recombination? Perhaps. But you are the experts.

The pursuit of science includes the ability to keep an open mind. In cases where results are diametrically opposite or very different, it is useful to examine the arguments of both sides or both sets of methods. Every method will have pitfalls. The important thing is not to be so attracted by the results as to forget to look at the pitfalls.

If one is not fully qualified to evaluate certain processes, it would be good then to read as many other experts as possible to see if there are consistent ways of approaching a problem. Some things that may appear obvious at first look may turn out to be artifacts on more systematic examination, or they could be accurate. You won’t know unless you ask yourself tough questions.

• Why do I think this person is right?
• What do other scientists say about this subject?
• Why are they saying what they are saying?
• What are the review processes that they undergo?
• If it is so straight forward that even I can see it, how come all these eminent scientists are not seeing it?
• Is it because they are stupid, stubborn, biased, corrupt, or (insert favorite description)…?
• Or could it be there is something wrong in my initial perception?

Sorry anon_22, I was just trying. I realize that I am probably not qualified. to evaluate these processes. :)

Under The Radar – at 16:52

You ask a very legitimate question. In fact, I have exactly the same question myself. The problem is, neither you nor I are molecular scientists, who can adequately qualified to comment on whether this method is valid or whether there are pitfalls that you and I do not know about.

LOL we posted at the same time.

Who among our little group here on this thread is qualified to speak to my post at 16:52, please? Thanks in advance.

lol. looks like you were quicker!

I am barely following all of this — and have this memory of a graphic that showed the genetic similarity of Homo sapiens to a banana. I’m gonna have to think on this . . .

LOL!

RBD – at 16:44

very few who are reading this thread will understand

The Great Minds are humble. If you think the discussion is above us, don’t post it. If you think not, then explain it.

The matches are EXACT. The recombination is OBVIOUS.

If it were obvious, the world would be agreeing. If all you can do is repeat “look at the slides” and type OBVIOUS and EXACT in caps, you’re wasting your time and ours. Why don’t you try gentle persuasion and teaching?

The scientists at the Vaccine meeting

Is there a list of the attendees?

I am happy to entertain this discussion a little further, but if you want to know why such discussions generally don’t get too far, look no further than your own post.

The floor is open.

But seriously, I am hoping someone can either validate what I have said in my post above, or correct me. I am not stating that recombination is the answer. I would not know. Unless my assumptions above are correct, that is.

I do hope I am not embarrassing myself by speaking without having a clue what I am talking about! LOL

disgruntled, The sequences go well beyond simple blast searches, which are useful for small regions of identity. The regions listed in the slides represent major portions of the gene.

Here is a walk through a coupel of the PB2 sequences

http://www.recombinomics.com/phylo/Canadian_Swine_PB2.html

53518 and 56626 are exactly the same for the first 550 positions (about 1/4 of the gene). 53518 the abruprty shifts to teh sequence from Korea. The rest of the geen is an exact match. 53518 has the forst 1/4 of the gene matching 56626 and the next 3/4 matching the Korean sequence EXACTLY.

56626 switches to the sequence in 23866 and that match is EXACT for over 1000 BP, which is about 1/2 of the gene. Those 1000 BP of identity match 11112 followed by North Carolina, then Tennessee, then North Carolina again. 56626 is an exact match with 53518 for the first 1/4, then 57561 for the next 1/2 of the gene.

It is these long stretches of identity with two more more other sequences that make the recombination obvious. Identity for 1/4 of the gene, followed by identity with another sequence for 1/2 or 3/4 of the gene is OBVIOUS recombination.

I like it best when we all play nicely together too, and I think we should all try.

UDR - I have decided to emulate your brave layman’s stance and enter the fray with some questions.

RBD: Thanks for your response to my vaccine question. So if we know what the parent sequence is, we can predict what the offspring sequence will likely be. Kind of like predicting what hair color a kid will have? That makes sense, and I do remember that Niman correctly predicted the S277N would show up in the Mideast. So if we know that sort of thing, then we could hypothetically make a vaccine for the projected offspring viruses/sequences ahead of time? It seems logical that if the S277N was predicted, that there must then be some way of making a vaccine to get ahead of it and if Niman has ideas on how to do that, I say let him flourish as it certainly can’t do any harm (and we don’t have a lot of other tools in our tool kit at this point).

I am interested in the science behind H5N1, but I am a layperson, so forgive the rudimentary questions. I thought that the existance of recombination was recognized and agreed upon - if not the extent or importance of it, but at least the fact of its existence. Is that not correct?

anon_22 - What methodologies are you looking for that would resolve what NS1 listed? Do people using different methodologies reach different conclusions regarding recombination?

From what I understand, the biggest dispute surrounding the idea of recombination is the disagreement over whether the changes arise sporadically by chance, or whether they are “programed” somehow (forgive the layman’s choice of words). Is there still a feeling that some of these recent changes can still be “one offs?” If so, wouldnt’ the way to solve this be to release as many full sequences as possible so that everyone can study them and the recombination/chance mutation debate can be put to bed? It seems like the sharing of full sequences would end the debate. Niman asks for the sequences to be released, so he sounds pretty confident (I can only suppose that he would not be so eager if he expected any of his theories to be disproven by their release).

If they did release full sequences, either a clear lack of recombination will be evident OR they may find lots more evidence of recombination than they ever suspected. Both results are maintained by their supporters, both points are debated (endlessly), and it seems that this could be resolved easily if full sequences were available to study. There does seems to be a lot of recombination being found. Or, if it is not recombination, what is it?

I suppose that the retaining of sequences could be just the way scientists go about protecting their intellectual property, their right to publish first, etc. But personally I worry that the sequestration of sequences has nothing to do with proving or disproving the recombination/random mutation debate and perhaps has rather to do with not wanting the bad news found within those sequences to get out. It makes me very nervous that partial sequences of very important full sequences are now being kept out of view. It makes me wonder why that would be done, and a quite possible answer could be that they may contain very bad news.

I was just reading my post at 16:52.”Sequences are made up of genes.” Funny! I get confused sometimes.

Under The Radar – at 17:25 I get confused sometimes.

Trust me, you are not alone. I’m right there with you!

Yeah Pixie! I agree!

Er, Pixie at 17:28, I hope you don’t think I meant I agree that you get confused! This is confusing.

I’m with Pixie.

RBD – at 17:06 Yes, I have seen the longer sections that match exactly. I was just describing the shortest usable stretch, and how anyone can try it for themselves. Niman appears to have a ‘favorite’ group of polymorphisms that he traces immediately whenever new sequences are released. If you read his polymorphism travelogues, you note that he reports them one segment at a time, for 2 to 7 segments. This pattern is repeated over and over. Thus, he appears to watch a fixed set of polymorphisms, spread over all segments. And he uses BLAST for these searches, either in short or long search mode. The long stretches of identity shown in the slides are the longest polymorphisms in his collection, but by no means the only ones he watches.

First of all, I appreciate everyone joining in with ideas and thoughts. This is constructive debate.

Secondly, identical sequences could also be the result of the virses being descendants of the same ancestor.

For example, if you look at this

You might say 48235 and 53518 has long stretches that are exactly the same. And you would be correct.

But what conclusion do you draw?

Is recombination, ie one virus acquiring this seqment from another, the explanation? Might there be other explanations?

Could these 2 sequences be descendants of the same ancestor, and along the way, some changes happened around positions 1931 and 2224, so that they now look different?

anon_22, did you mean 48235 and 55383?

DemfromCT wrote:

“Do so. OTOH, anyone’s banning is the prerogative of the mods and not up for discussion, so keep your focus. Should it stray on to the topic of banning, the thread will be closed. Is that crystal clear?”

I’m not sure, but I think that Dem is saying that having the power to censor is the same as having the authority, and that anybody that speaks against the moderators will be censored. That is way sick. What isn’t clear to me is what kind of people would submit to such bufoonery.

It doesn’t take any courage for me to speak against you “people”. You are cowards hiding behind fake names.

It’s not our sandbox.

Back to the very interesting discussion on vaccines and sequences…

Or they could be the result simply of stretches of invariable codons in a gene.

For example, in this paper Long term trends in the evolution of H(3) HA1 human influenza type A, Fitch et al, PNAS, they examined 254 HA genes from H3N2 isolates from 1984–96, and constructed a phylogenetic tree.

 Now the HA gene is one of the faster changing genes, being subject to human immune responses.

Of these, depending on where they are placed in the tree, no of variable codons (out of 329) for those on the ‘trunk’ of the tree is only 47 (18%), whereas it’s far higher at the ‘tip’ is 175.

So we can see that even for HA, depending on what strain you get, it is possible to have 82% of codons unchanging in a large sample.

yes. LOl

pix,

this forum should be renamed chickenflutalk

Remember, a new theory is only correct if it cannot be explained by other simpler solutions.

so says a nameless coward

anon_22 - at 18:06

Does the simpler solution offer the predictibility that Niman’s interpretation of recombination offers? That seems to be what makes his concept different.

and according to actual expets, occam’s razor is to distinguish between theories that are otherwise equivalent, not merely those that explain but those that argue

Pixie – at 18:08

anon_22 - at 18:06

Does the simpler solution offer the predictibility that Niman’s interpretation of recombination offers? That seems to be what makes his concept different.

Yes, but does it test out in reality? Remember that if you make a lot of predictions, then some of them might happen. Again, in order to answer that question, one would need a more systematic way of measuring what exactly were his predictions, how exactly were they derived from his theories, and how many times did they turn out to be right, and, most importantly, whether this ‘prediction success’ could not be explained any other way, such as chance?

It’s more subtle than just the long stretches in two sequences. It’s that the long sequences appear cut into other sequences in various combinations, without error, for over 25 years. And the changes at position 1931 aren’t random changes, they are cut in as identical copies of a stretch of an Ontario sequence. You have to explain both the fact that the long sequences are identical from 1977 to 2004, and that the pieces of the long stretches contain identical sections, and that there are other sequences, notably in 53518 and 11112, that occupy the same spot with different coding, that do the same thing as the Tennessee segment, so you can’t explain it by saying it’s an ‘invariant’ region. This picture is not consistent with random mutation; it looks more like hybridization. And that implies dual infections.

Pixie – at 17:21

If so, wouldnt’ the way to solve this be to release as many full sequences as possible so that everyone can study them and the recombination/chance mutation debate can be put to bed? It seems like the sharing of full sequences would end the debate. Niman asks for the sequences to be released, so he sounds pretty confident (I can only suppose that he would not be so eager if he expected any of his theories to be disproven by their release).

When FAO, followed by Indonesia, announced the release of sequences, the Niman supporters abruptly ended some of the debates. It was a really strange silent period. I was thinking we will hear more in a few weeks from Niman about what he found out of the released sequences. If recombination is so OBVIOUS CLEAR DATA (all Niman followers favorite terms), those additional sequences should validate some of his predictions. I have not heard any more findings about Indonesia sequences.

I suppose that the retaining of sequences could be just the way scientists go about protecting their intellectual property, their right to publish first, etc. But personally I worry that the sequestration of sequences has nothing to do with proving or disproving the recombination/random mutation debate and perhaps has rather to do with not wanting the bad news found within those sequences to get out. It makes me very nervous that partial sequences of very important full sequences are now being kept out of view. It makes me wonder why that would be done, and a quite possible answer could be that they may contain very bad news.

I have the same fears as you. I can’t determine how selfish or corrupt ‘eminent’ scientists may be to draw that conclusion. Let’s just say that your second scenario is correct i.e. the really bad news of recombinants are found in the sequences hidden from public view. If that were the case, I would expect secret deals cut between all these governments and Dr. Niman on the one hand to make ‘predictor’ vaccines, and I would also expect these ‘corrupt’ scientists jumping on the bandwagon and obtain their own funding for recombination research. These do not seem to have happened. I don’t think such a break through in science can be suppressed. If it’s so hot, I would expect a black market on Niman-Inside vaccines, even if the governments suppress them.

Quoting Niman:

Analysis of donor sequences in the Middle East identified donor sequences on H9N2, which is endemic to the Middle East. Dual infections involving Qinghai H5N1 and H9N2 endemic in the Middle East, could generate S227N via recombination. Therefore a warning was issued on October 22, 2005. At that time, no human H5N1 infections involving the Qinghai strain had been reported.

In January, human H5N1 cases were reported in Turkey and H5N1 isolated from the index case was the Qinghai strain with the S227N acquisition. Although S227N was not found in an isolate from the sister of the index case, it was found in a second human case from Turkey. Thus, of the four human sequences released from Turkey, two had the S227N change. WHO issued an update Febraury 20, 2006 indicating S227N had not been fixed in the Qinghai strain, but the number of human sequences released was limited to one case from Egypt, Iraq, and Djibouti. Subsequently the sequence from one human case from Azerbaijan was also released.

================================== This is a specific prediction based just on analyzing travel paths and figuring out which donor sequence had S227N. Niman subsequently showed that the ‘child’ strain had inherited many polymorphisms from the ‘parent’ strain, not just S227N, so its presence wan’t just a fluke. The new strain was indeed the hybrid he predicted.

anon_22 – at 18:13 Yes, but does it test out in reality? Remember that if you make a lot of predictions, then some of them might happen. Again, in order to answer that question, one would need a more systematic way of measuring what exactly were his predictions, how exactly were they derived from his theories, and how many times did they turn out to be right, and, most importantly, whether this ‘prediction success’ could not be explained any other way, such as chance?

I completely agree, although Niman might say that the sequestration of the sequences hampers his work, particularly this part of it. I also wonder if a portion of this work might not be covered in his patent.

Now is the time for everybody to pull out the stops, though, and release sequences and get to work on building the kind of airtight theories around them that you describe, particularly if predictibility is a possibility. Nothing else makes sense. Releasing key sequences too late, or proving that something could have been predicted too late, just cannot be part of our hindsight.

Pixie,

I agree that sequences need to be made available for urgent research.

But what we are debating here is a more fundamental question, is recombination a significant mechanism for evolution of influenza A viruses? This question can be answered by studying not just H5N1, but all the seasonal flu sequences that are in the database. For example, the study that I quoted in 18:01 used 254 sequences from GenBank, and came up with really valuable information.

So let’s be careful that the issue of disclosing H5N1 sequences does not become the smokescreen for inadequate explanation of recombination as a theory.

Notice I am not saying that recombination is NOT a significant mechanism. I am saying that the evidence for it is insufficiently elucidated.

There may come a day when it will. Or not.

ANON-YYZ – at 18:14 I would also expect these ‘corrupt’ scientists jumping on the bandwagon and obtain their own funding for recombination research. These do not seem to have happened. I don’t think such a break through in science can be suppressed. If it’s so hot, I would expect a black market on Niman-Inside vaccines, even if the governments suppress them.

Very interesting ideas.

They are doing a pretty good job of supressing those sequences, though. A very impressive job, under quite a lot of extraordinary pressure in extraordinary times. A pandemic is in the offing, humanitarian appeals have been made, and still data on those sequences is supressed. That does make me wonder if other scientific knowledge is supressed. It might not be, but it has to make one wonder.

Your idea of a black market vaccine is a possibility, although I think that it is more likely here that Kuhn’s paradigm shift is at work instead, and that takes time, and engenders a lot of resistance that eats up even more time. I find that explanation more likely than that of corrupt scientists. So I’d look to Kuhn, then, not corruption, on that front.

I would suggest that anyone interested in recombination (or any subject, for that matter) to please read as widely around that subject as possible. There’s a reason why modern science is based on multiple references, peer review, control study, statistically significant methods, and detailed description of methodology for others to scrutinize. It is actually rather similar to the ‘hive mind’ we talk about here.

The more brains working on one problem, the more likely you will get closer to the solution.

I would suggest at least having a quick read of the references I gave (and I’m still working on some others), especially the free ones, or any other references you can find on this topic. You may not find answers laid out exactly the way you want, but sometimes just going through how other scientists approach a problem can be very instructive.

anon_22 – at 18:31 Notice I am not saying that recombination is NOT a significant mechanism. I am saying that the evidence for it is insufficiently elucidated.

There may come a day when it will. Or not.

Fair enough. May we be granted the time to find out.

The predictive possibilities implied by recombination are so potentially important, considering the situation that we are facing, that we need that edge if we can get it. I’d be very happy if we find ourselves here, still arguing about it, situation unchanged, five years from now. Yes I would.

disgruntled – at 18:13

It’s more subtle than just the long stretches in two sequences. It’s that the long sequences appear cut into other sequences in various combinations, without error, for over 25 years. And the changes at position 1931 aren’t random changes, they are cut in as identical copies of a stretch of an Ontario sequence. You have to explain both the fact that the long sequences are identical from 1977 to 2004, and that the pieces of the long stretches contain identical sections, and that there are other sequences, notably in 53518 and 11112, that occupy the same spot with different coding, that do the same thing as the Tennessee segment, so you can’t explain it by saying it’s an ‘invariant’ region. This picture is not consistent with random mutation; it looks more like hybridization. And that implies dual infections.

True. However, scientific understanding of the basis of viral evolution is shall we say still pretty rudimentary. We don’t really know why certain changes are more likely to happen, except to say there may be selection value. It is possible that on top of single gene changes, there are complex combination changes that are selected. This is only just now beginning to be studied, with the availability technologically of studying large numbers of whole genomes.

There are a lot of things we do not understand still. So one can propose recombination as a theory to explain what we do not understand. Which I can accept. But we have to be careful and not act as if it is proven. Because if we do, our decisions may be directed incorrectly. And, we stand in danger of closing our minds to other possibilities. Which will spell the death of pursuit of knowledge.

Anon_22,

The data in slide 26

http://www.recombinomics.com/phylo/Canadian_Swine_PB1.html

used 1109 human sequences to show how human seasonal flu evolved via recombination.

Pixie – at 18:33

Pixie – at 18:40

Paradygm shift may be at work, but we shouldn’t wish it to be the case because we need it so badly.

Hope is not a plan.

RBD – at 18:48

Anon_22,

The data in slide 26

http://www.recombinomics.com/phylo/Canadian_Swine_PB1.html

used 1109 human sequences to show how human seasonal flu evolved via recombination.

Actually, I read it, and didn’t get how that implies recombination at all. Can you explain?

anon_22 – at 18:28

But what we are debating here is a more fundamental question, is recombination a significant mechanism for evolution of influenza A viruses? This question can be answered by studying not just H5N1, but all the seasonal flu sequences that are in the database.

I also agree that all H5N1 sequences should be released.

Have the seasonal flu seqnuences been referenced by Dr. Niman? The delay of H5N1 sequence release should not be a factor impeding the validation of his theory then.

anon_22, can you help get the sequences released? I mean, do you have any influence with the people who are withholding the sequences?

Or, are there many people from the WHO member states that you could join with who could collectively demand that the sequences be released?

I just don’t understand who is telling the WHO not to release the sequences. Surely someone must be insisting that they don’t release them. But who could that be?

In fact, I think we should find out who these people are and put their names right here on this forum.

Under The Radar – at 19:06

WOW, you are according me more power than I can possibly imagine!

No, I’m a nobody in that world of high politics. I count myself very lucky if some of those high and mighty would deign to read what I write every now and then.

LOL

In fact, I think this should be in the mainstream media, so the people can call for their government leaders to demand that the sequences be released.

I am quite sure that if the people knew that scientists need these sequences to do their research to develop a vaccine to try to avert a pandemic that could kill upwards of two billion people on this planet, and that there were people who ensuring that these sequences were withheld from the scientists (for WHATEVER reason. there is no reason or valid excuse), ANY scientists, the people would be mad as h*ll about it and would demand that the sequences be released!

Yep, the story about the withholding of the sequences, why we need them, and the names and faces of the people responsible for the withholding of the sequences right there on TV and in the papers.

anon_22 – at 18:48

I have read many of the reports you cite, and I have also posted critiques of their methods on at least two occasions on this forum. You have repeatedly reminded me to keep an open mind, and I have done this faithfully. And after doing a lot of homework, and asking many questions, and doing my own analysis, I do not find the random mutation paradigm explains the facts as shown in the sequences. I understand the sequence database is biased, in particular, it contains mainly the sequences that the hoarders don’t consider very important. It is also biased because the isolation methods used select against even detecting multiple infections, they bias against finding avian polymorphisms in mammal cell culture, mamallian polymorphisms in avian cell culture, and a pile of other flaws. It’s the best database we’ve got.

I have patiently awaited Webster’s predicting anything. I’m still waiting. But I’m not predicting he will. People who believe in random mutations don’t make predictions, which itself is a prediction.

A year and a half ago, I might have agreed with you. Now, based on events and an extensive review of the evidence, I don’t. I am perfectly willing to be pursuaded otherwise, but so far the competing evidence is not convincing.

I wonder if there is anyone in the mainstream media brave enough to actually do that.

Under The Radar – at 19:27

it’s actually been in the media, especially the science media. Nature especially has run with this, and so has Nicholas Zamiska at the WSJ with a major feature on Ilaria Capua and a more recent one on Henry Niman. ‘Nature’ has run signed letters from many world-renowned scientists, as has ‘Science’.

We’ve pushed it in various venues besides here (this was from July and placed it in the political arena). It’s not new, has gotten some results and is an ongoing issue.

btw, the new rumors thread has some discussion of sequence release.

disgruntled – at 19:26

Fair enough. We shall probably not find satisfactory answers for a little while. Let’s both (all) keep looking, though.

BTW I doubt that Webster is into prediction, though. Not very explicit ones, at least.

DemFromCT – at 19:46

Thanks Dem. Then I wonder why the averge person on the street doesn’t know anything about it. Maybe because most people don’t read Nature, Science or the WSJ. They watch Oprah.

1. most people don’t read the science news
2. most people don’t think there will be a pandemic
3. most people don’t understand why sequeence release is a big deal
4. most people would side with WHO if they got past 1–3.

I can understand editors not going with this.

Dem, My point was, why don’t they put it on Oprah, if that’s what the people watch. (Oprah is just an example.)

They put Mike osterholm on Oprah to tell the people about the bird flu. Why doesn’t he go on there again and tell everyone about the WHO hoarding the sequences, and why we need them?

If the people were told the truth, they would understand why the sequence release is a big deal. I do not believe they would side with WHO if they knew the truth.

I believe there would be some pressure brought to bear on the PTB responsible for hoarding the sequences.

So what shall we do? Lament the fact that we don’t have the sequences? Try to think of all the reasons why we can’t get the sequences? Is there nothing more we can do?

I’m just saying…

Sorry, RDB, I hope I didn’t hijack your thread, and I sure didn’t mean to if I did. I just get so irritated about this debate over is it recombination or isn’t it, when the real issue is the hoarding of the sequences. If the sequences were released, there would be no more arguing about it and maybe some real progress could be made.

Just got home from work and am many hours behind on this thread, so forgive the late postings, but…

RBD at 15:20 “This was done before H5N1 flew into the Middle East. The H5N1 could acquire S227N by recombining with H9N2, which was endemic in the Middle East. The recombination could be predicted down to the specific nucleotide that would be acquired.”

This statement made me start to contemplate why/how Niman made the prediction for this particular change? What was it about this sequence that made it so susceptable to recombination that he could predict the change “down to the specific nucleotide?”

AND Anon 22 at 18:48 “True. However, scientific understanding of the basis of viral evolution is shall we say still pretty rudimentary. We don’t really know why certain changes are more likely to happen, except to say there may be selection value. It is possible that on top of single gene changes, there are complex combination changes that are selected.”

More of the same thought provoking question. Are their certain areas in a gene more likely to be cut and switched than others? Is this part of where Niman gets his predictions from? It is one thing to simply predict that a recombination of SOME PART of the genes of one virus with another will occur, but to be so specific? Would these predictably recombinant sequences have to do with their location near intron sequences on a gene?

The prediction, made October 22, 2005

please ignore that last little bobble line, inadvertantly copied and pasted.

Mary in Hawaii – at 21:44

Thank you for saving the thread. I was so afraid I had killed it and I was feeling pretty bad about that.

so, what’s “hybridization” here and how does it explain streches of identity and why is double infection necessary ?

In this debate about recombination vs other mechanisms of mutation, the debate seems to divide us into two groups: those who focus on the length of the identical sequences that are cited as evidence of recombination, and their wide distribution along the flu genome; the other group recognizes the homology but questions methods of alignment, identification, or whether other explanations might not also account for the data.

For the sake of moving the discussion forward, let’s just say that both arguments have their merits. Then, as a research question, it would be useful to robustly test alternative hypotheses. As a practical matter though, it is useful to focus on the different implications of the two mechanisms of mutation. In reassortment, the probability of the emergence of a human pandemic strain of H5N1 is low, the rate of mutation is slow, and the probability of dead ends is high. With recombination, the probability of a pandemic strain emerging is much higher, the rate of mutation is higher, and the probability of dead ends is lower, since “working fragments” are being swapped. So the important point here is that if you choose for a moment to believe that recombination is driving mutation in H5N1, then you are confronting a reality that is more dire than conventional wisdom would have us believe. Because the data at the very least don’t rule out recombination, then I think it makes more sense to prepare as though recombination were right, rather than as though reassortment were right, because in the former case, you’ll blow some money but be ready for the worst case, while in the latter case, you’ll be caught flat-footed. Also, importantly, recombination makes predictions, and we need to be able to make predictions in order to begin developing vaccines now for a virus that will be upon us in 6 months. Since reassortment doesn’t allow prediction (please correct me if I’m wrong on this).

A speculation: From a natural selection standpoint, if reassortment and recombination are both occurring, it seems to me that natural selection would favor mutation by recombination, because mutations would lead to the incorporation of sequences that code for proteins that are functional and have conferred selectional advantage to the carrying virus, whereas reassortment allows for the emergence of qualitatively new viral machinery, but the probability of the emergence of new functional and adaptive proteins is low. If this is true, then natural selection should favor viruses in which mutation occurs by recombination.

One thing in closing. There has been much talk about how science progresses, along the lines of the Kuhnean paradigm shift, etc. In practice, if you have theories or data that challenge the dogma, your chances of getting the data published are much lower than if you support the dogma. My experience with scientists is that they are extremely averse to trying new methods, or contemplating new ideas. I can understand why Niman got fed up with the whole peer review nonsense. Peer review filters out a good bit of junk science, but it also can act as a formidable barrier to the emergence of new ideas. There is no scientific manuscript on this earth that can survive a hostile reviewer, and all you need is one to keep a paper from being published. We live with it for the same reason we live with democracy, because it is the best of a series of lousy choices. In my opinion though, it decimates many of the best ideas from science. That’s why I am deeply offended by the “banishing” of Niman from a discussion that he is perhaps best qualified to engage in.

Recombination in action. Human H1N1 acquiring swine sequences, as well as 1918 pandemic sequences via Canadian swine (real data from real people)

http://www.flutrackers.com/forum/showthread.php?t=12642

CY013580 A/Otago/5/2005 PB2 (1) 2283 2005 H1N1

CY016682 A/South Australia/55/2005 PB2 (1) 2283 2005 H1N1

CY016690 A/South Australia/56/2005 PB2 (1) 2293 2005 H1N1

CY016698 A/South Australia/57/2005 PB2 (1) 2295 2005 H1N1

CY013604 A/Waikato/14/2005 PB2 (1) 2287 2005 H1N1

CY013564 A/Wellington/10/2005 PB2 (1) 2280 2005 H1N1

CY014014 A/Wellington/11/2005 PB2 (1) 2282 2005 H1N1

CY013572 A/Wellington/12/2005 PB2 (1) 2293 2005 H1N1

CY013596 A/Wellington/13/2005 PB2 (1) 2285 2005 H1N1

CY015587 A/Wellington/14/2005 PB2 (1) 2283 2005 H1N1

DQ280229 A/swine/Ontario/48235/04 PB2 (1) 2339 2004 H1N2

DQ280205 A/swine/Ontario/55383/04 PB2 (1) 2280 2004 H1N2

CY004946 A/mallard/Alberta/30/2001 PB2 (1) 2341 2001 H4N8

M73513 A/Swine/Tennessee/24/77 PB2 (1) 2341 1977 H1N1

CY009923 A/swine/Tennessee/25/77 PB2 (1) 2309 1977 H1N1

CY009635 A/swine/31 PB2 (1) 2313 1931 H1N1

M55469 A/Swine/Iowa/1976/31 PB2 (1) 2326 1931 H1N1

M73515 A/Swine/Iowa/15/30 PB2 (1) 2341 1930 H1N1

DQ208309 A/Brevig Mission/1/1918 PB2 (1) 2280 1918 H1N1

NS1 - I misread your earlier post. I read the thread you mentioned, searching for a reference to the quasi-species model for context. I understand now what you meant.

ducksoup – at 22:20

Re: your 2nd paragraph

The problem is all manners of Witch Doctors can use the same argument to sell snake oil. In fact, it is not unknown for terminal cancer patients and their families to give their life savings to some one who claim to have alternative therapies. If it fails, it’s god will. At least we have tried out best.

Re: your 4th and last paragraph

Niman’s theory is being represented quite well here by RBD, with an almost identical writing style. I was asked to start on topic to discuss the slides presented at the Vaccine Summit in Boston in August. which started this thread.

Nothing stops Dr. Niman from publishing his own book, even a PDF, on his own website. The only time I hear about him is when there is some flu news, and his disciples hyped up the news so people get bad dreams from his innuendo.

I just don’t buy the notion that all other scientists are close-minded, corrupt, self-serving, pro-establishment, can’t handle paradygm shifts. I am running out of adjectives.

Why is it always every body elses’ fault with so much hostility? If one can’t get other scientists to become allies, may be, just may be, sour grapes grow on junk science.

ducksoup – at 22:20

Very nice summary and thank you. For those that don’t know which way or whether both ways of viral evolution occur, prepping makes sense (especially because we know that pandemics happen). It is unfortunate that Webster and Taubenberger and others aren’t all here to debate, but such is life.

For those who do not feel that recombination is the way that viruses evolve, there is still a need to prep.

Robert G. Webster, Ph.D., and Elena A. Govorkova, M.D., Ph.D.

My point is that there is more agreement than this discussion would make it seem ;-)

Anon-yyz

Except for the last sentence of my post, I didn’t refer to Niman, I talked about his ideas. I’d be interested in reading your responses to the issues I raised, but since you left that out of your post, I just want to clarify: I like RBD’s writing style, but I’m interested in Niman’s ideas, so I’d rather have him in the discussion. I’m not his disciple, nor are those who basically stick to the data and find that it supports Nimans theories.

You misinterpret or misrepresent what I wrote about scientists. In the main they are pretty closed-minded. I don’t know what you do for a living, but if you were a scientist, I don’t think you’d find that notion so controversial. While there are plenty of exceptions to the generalization, because in general there is a resistance to new ideas, it is difficult to get them disseminated. That’s very different than saying that all other scientists are close-minded. As for corrupt, do you have other words to describe the lack of disclosure of gene sequences? Note that it is a minority of virologists who are making a stink about this, and not even Nature is being transparent.

Please, I’m not interested in discussing Niman. I’d like to hear from Niman on these issues. Barring that, I’d like to discuss the issues.

Mary in Hawaii – at 22:46 NS1 - I misread your earlier post. I read the thread you mentioned, searching for a reference to the quasi-species model for context. I understand now what you meant.

THE ABOVE POST WAS NOT FROM ME. I AM MORE THAN A LITTLE AGGRAVATED THAT SOMEONE WOULD POST SOMETHING PRETENDING TO BE ME!! Not that what they posted was of any significance, but what the h*ll is going on here? This is supposed to be a serious forum comprised of thoughtful and intelligent individuals trying to find solutions to a problem which may be one of the worst crises mankind has ever faced, and someone is playing games with it. Has anyone else out there run into this problem?

The post in question is now attributed to Sock Puppet. - pogge

LOL!

ANON-YYZ – at 23:22 Your post to Duck soup was incredibly hostile for no reason whatsoever. You ascribe things to him he never said, and make claims about “hostility” when his tone was very balanced and calm, and his content very open-minded and even handed. Look in the mirror.

That unwarranted attack, directly following the faked “post” ascribed to me which I never wrote, makes me wonder if there are people here purposely trying to breed mistrust and animosity for some nefarious purpose. We have bigger things to worry about here. Let’s move on.

lol lol!

NS1 - I am still an idiot.

Mary in Hawaii – at 00:06

Since your comment about my post is followed by your remark about the fake post, I have to ask the mods to identify who posted that. It wasn’t me.

LOL! LOL! LOL!

I just banned a sock puppet. Give me a second to sort this out.

The numerous posts from Under The Radar, two from Mary in Hawaii and several from anonymous all came from the same IP address. That address is now blocked from posting. I’ll edit the thread to indicate which posts attributed to MiH appear to have come from someone else.

I only see one from Mary in Hawaii that was not from me, the one at 23:55. sigh. But thanks for clearing the lines.

PLEASE SKIP THIS POST IF YOUR ONLY INTEREST IS IN THE SCIENCE

ducksoup – at 23:51

I may have read your particular post in the larger context of what you have previously written through out this thread, and your strongly worded posts in the thread about why Niman was banned. We both have on-line personalities, and some times it is easy to read more context and meaning into what was said. Since you asked, I will tell you I am neither a scientist, nor work in the health care profession. I am not a PR executive, promoter, investor nor do I work in any field that puts me in a position of preferring one scientific theory over another as far as recombinomics is concerned. I am simply a prepper with a deep sense of wanting to know the truth. As many have said, the stakes are too high to be partisan. As a general stance, I am against sensationalism or excessive promotion. I am not qualified to comment on the technical details of the theories. I am simply reading with interest the debate of the science, to afford me a better assessment of the situation or risks of pandemic.

Let me apologies to you if you feel hurt, that I misinterpreted your writing.

If I understand correctly, you do not seem to have an issue with my comments about your paragraph 2.

I wrote:

‘’I just don’t buy the notion that all other scientists are close-minded, corrupt, self-serving, pro-establishment, can’t handle paradygm shifts. I am running out of adjectives. Why is it always every body elses’ fault with so much hostility? If one can’t get other scientists to become allies, may be, just may be, sour grapes grow on junk science.’‘

As far as my comments referencing your paragraph 4 is concerned, I was really saying that I cannot believe Niman theory’s lack of acceptance is a result of all other scientists being at fault in one form or another. While you view scientists as in the main they are pretty closed-minded, I cannot believe that ALL of them are closed minded, and you just agreed with me on that. That being the case, do you agree that Niman’s recombinomics should have at least got more acceptance by other credentialed scientists? One almost have to argue every one else is close-minded, corrupt, self-serving, pro-establishment, can’t handle paradigm shifts to explain (as I understand it) the lack of acceptance of Niman’s theory by other scientists. One cannot explain, where the subject matter of recombinomics is brought up, the amount of shall I say unusual forum behavior not attributed to people who don’t buy the theory.

Anon YYz

There are plenty of precedents where obscure scientists have been heaped with ridicule by the mainstream for proposing a theory so at odds with the dogma as to be deemed crazy, only to be proven right. Prions came out of left field, so did heliobacter (off the top of my pretty sleep-deprived head). So yes, I think it is perfectly possible that Niman is right and everyone else is wrong. It sure looks that way. They aren’t necessarily self-serving, just wrong.

ducksoup – at 02:56

Precedents or not, Niman is not one of those named historic figures. Associating him with these examples doesn’t turn him automatically into those examples, and thereby removing the need for scrutiny. No one has excluded the possibility Niman’s theory is right, which is why this forum has a track record of allowing discussions on the subject matter.

It is always possible for Niman’s theory to be right. It just needs to be proven by the proponent of the theory. Time and again, when asked specific question, the Niman supporters will turn the question around, repeat the OBVIOUS CLEAR DATA mantra, and ask the querier to prove recombinomics wrong. To me, that is a dead give away that the proponents of recombinomics know there are flaws in the theory, and cannot answer the questions.

If recombinomics is so right, there is no need to be evasive, there is no need to quote analogies, there is no need to call existing scientific theories by the name of dogma, or any other names. All it takes is to self publish a scientific paper on Niman’s website, and have a forum there to allow questioning of the methodologies being used to establish the proof. All other scientists submit to that standard. Why should the rest of the world accept a lower standard of validation and accountability?

You wrote:

So yes, I think it is perfectly possible that Niman is right and everyone else is wrong. It sure looks that way. They aren’t necessarily self-serving, just wrong.

This style of proclamation without proof has been presented time and again by Niman proponents. I agree with the first sentence. I don’t agree with the second and the third. Just calling all existing scientists wrong doesn’t logically turn the proclamtion into the truth.

Structuring the sentences the way you did intentionally or not, unfortunately, does have an effect on the brains of most people, who will subconciously accept the 2nd and 3rd sentences as truisms without logical analysis, after turning on the green light for the first sentence.

Calling every one else wrong doesn’t make every one wrong. It certainly won’t prove Niman right. By avoiding scrutiny, the message I receive from you is that there is no substance in the theory, only hot air.

Recombinomics may be interesting, even entertaining, just like astrology. However, I can’t take it to the bank yet, especially when my family’s lives depends on it. I submit that’s why no one (to my knowledge) has yet put money on the table to make a Niman-Inside vaccine.

Anon-YYZ,

Will you allow Niman free usage of your coinage, Niman-Inside, if Recombinomics does prove satisfactory?

I think its very clever. I still don’t understand the source of the powerful emotion behind your repeated, vindictive affronts? Do you believe that sarcasm makes casual personal attacks kinder or more stimulating to the discussion?

NS1

I’d be grateful if you could look at what I wrote at 22:20 above. Specifically, is it reasonable to infer that recombination accellerates the process by which a pandemic strain will emerge? Second, doesn’t it make sense to argue that if both reassortment and recombination are occurring in parallel, natural selection will favor recombination?

Anon-YYZ do me a favor: ignore me and my posts. The french have a saying: “when you’re dead it’s for a long time, but when you’re a fool it’s for ever”. You’re wasting your time trying to enlighten me. Please help others.

ducksoup – at 22:20

Thank you for your efforts in keeping this topic on track. We are here to discuss the science, not personal feelings.

As a practical matter though, it is useful to focus on the different implications of the two mechanisms of mutation. In reassortment, the probability of the emergence of a human pandemic strain of H5N1 is low, the rate of mutation is slow, and the probability of dead ends is high. With recombination, the probability of a pandemic strain emerging is much higher, the rate of mutation is higher, and the probability of dead ends is lower, since “working fragments” are being swapped.

With respect, I think you may be confusing some terminology here. Reassortment and recombination are not 2 mechanisms of mutation. In fact, neither of them are mutations at all. Mutation happens when a specific gene coding for an amino acid, or codon, is changed during viral replication. This happens frequently with RNA viruses because of their inability to ‘double-check’ and correct mistakes.

Reassortment happens when a whole strand or gene segment, containing all the codons, is swapped with another virus. This happened in the 1957 and 68 pandemics. Recombination is when sections of a gene sequence, containing a number of codons but not the whole segment, is swapped.

Potentially all these mechanisms can produce a pandemic strain.

So the important point here is that if you choose for a moment to believe that recombination is driving mutation in H5N1, then you are confronting a reality that is more dire than conventional wisdom would have us believe.

Possible. Although there is a upside and a downside to that. When people are repeatedly alarmed and the event that they fear didn’t happen, it may breed complacency and disbelief of everything else that comes after.

Because the data at the very least don’t rule out recombination, then I think it makes more sense to prepare as though recombination were right, rather than as though reassortment were right, because in the former case, you’ll blow some money but be ready for the worst case, while in the latter case, you’ll be caught flat-footed.

I think we should prepare for worst case irrespective of whether we believe in recombination, because we do not have a lot of historical guidance as to the speed with which a pandemic virus can arise, or the scientific understanding to know what exactly the requirements are for a pandemic virus. I mentioned this in passing in the ‘dummies’ thread, that receptor binding is just one element of many, most of which we have not been able to elucidate at all. I’m working on a couple of other publications after which I’ll start a topic on that.

Also, importantly, recombination makes predictions, and we need to be able to make predictions in order to begin developing vaccines now for a virus that will be upon us in 6 months. Since reassortment doesn’t allow prediction (please correct me if I’m wrong on this).

Well, recombination actually does not make predictions, if you read the literature on recombination. It is Niman who claims he can use this for predictions.

A speculation: From a natural selection standpoint, if reassortment and recombination are both occurring, it seems to me that natural selection would favor mutation by recombination, because mutations would lead to the incorporation of sequences that code for proteins that are functional and have conferred selectional advantage to the carrying virus,

Or functional units arise because selection pressure allows certain combinations of mutations to be more frequently selected than others. There is some preliminary work on seasonal flu, in the NIH Whole Genome Sequencing project.

I think the big unfortunate mistake in virology is the continuing use of the word ‘random’ when describing mutations. While mutations when they arise at the molecular level may be random, the advantage or disadvantage that any mutation confers will automatically ensure that the mutations that we actually find on strains that survive are far from random. Selection of ‘functional’ mutations or collection of mutations would favor the retention of certain changes over others.

whereas reassortment allows for the emergence of qualitatively new viral machinery, but the probability of the emergence of new functional and adaptive proteins is low. If this is true, then natural selection should favor viruses in which mutation occurs by recombination.

Not true, since reassortment happens with a whole gene segment from existing ie fit and surviving, viruses.

One thing in closing. There has been much talk about how science progresses, along the lines of the Kuhnean paradigm shift, etc. In practice, if you have theories or data that challenge the dogma, your chances of getting the data published are much lower than if you support the dogma. My experience with scientists is that they are extremely averse to trying new methods, or contemplating new ideas. I can understand why Niman got fed up with the whole peer review nonsense. Peer review filters out a good bit of junk science, but it also can act as a formidable barrier to the emergence of new ideas. There is no scientific manuscript on this earth that can survive a hostile reviewer, and all you need is one to keep a paper from being published. We live with it for the same reason we live with democracy, because it is the best of a series of lousy choices. In my opinion though, it decimates many of the best ideas from science. That’s why I am deeply offended by the “banishing” of Niman from a discussion that he is perhaps best qualified to engage in.

Well, as someone suggested, it is possible for a maverick scientist to not go through the mainstream process but still give a thorough account of his methodology and results, so as to allow others to evaluate it properly. If you look at published papers, and look at the thoroughness with which scientists are required to document what they are doing, it is very hard to think that just taking someone on faith (aka when they say it is OBVIOUS) is better.

Plus, the peer review process allows for anyone who disputes the findings a proper place to raise doubts, objections, rebuttals, and the original author to defend, clarify, or add supplemental information. It’s not perfect, but it is not such a ‘black box’ operation as some would suggest either.

In addition, recombination is not a new idea. It is a well accepted mechanism of change for many other viruses and bacteria.

What is in dispute is whether it happens to a significant degree in influenza A viruses. That of course, turns it into a dispute of methodology, not ideology.

In other words, it is not a matter of whether you believe in recombination as a process. I do, for example.

It is a matter of comparing the reasoning of those who say that it happens frequently and is an important mechanism to those who say it is not. That’s why it is important to compare and evaluate the processes by which the different parties arrive at their conclusions. Hence the focus on methodology.

I’d like to ask again a question I posted yesterday: Are their certain areas in a gene more likely to be cut and switched than others? Is this part of where Niman gets his predictions from? It is one thing to simply predict that a recombination of SOME PART of the genes of one virus with another will occur, but to be so specific? Would these predictably recombinant sequences have to do with their location near intron sequences on a gene?

Hello,

I have never posted at Flu-Wiki before, but I think it is useful to have this discussion on the predictability of a pandemic vaccine based on recombination. From what I understand of Niman’s method he queries a database for specific sequences focusing mostly on receptor binding sites and seeks close and exact matches to his sequence query. Based on the fact that he can find these sequences he assumes that this proves recombination rather than what I think may be mutation followed by geographic spread. I do agree that his data does not rule out recombination. As a molecular biologist I just don’t think it proves it. I have to agree with most of Anon’s comments on the validity of this blast searching method for proving recombination. I have asked Niman on a different forum of whether or not he has ever run any of the computer programs which can validate the presence of genetic recombination based on sequence and phylogenetic data. I have also asked whether or not any test tube experiments of co-viral infection have ever proved this theory out. I have received no response from Niman as to these specific questions. I also agree with the observation that it will be unlikely that we will ever have sequence data on two or more variants from the same host that would be necessary to prove this theory out unless sequence data was provided after a recombination experiment was carried out in a test tube controlled environment. I also think Mary in Hawaii asks a great question as to what dictates areas of recombination or copy choice by a RNA polymerase. Are there signals that dictate where a polymerase will jump. How does Niman know this a priori? Just food for thought.

Curious

Curious – at 21:50

Thank you for posting. There are more questions than answers for many of us, alas, but i appreciate your lending us your expertise and posting.

In the past, have virologists looked at the polymorphs on IA before this way?

Is it unknown if changes to the virus happen in very small steps?

Can very small steps lead to a big change in the virus and cause it to become a pandemic strain?

Do you need a big change or just small changes in the right points to become a pandemic strain?

The idea of recombination makes a whole lot of sense to me. And watching it play out over the last 1 1/2 years has seemed to confirm the idea of it. A recombination event, the fixing of certain recombinations, infectious spread amongst a population, the migration of that population. And that fixed recombined virus again recombines with other viruses it meets along the way.

One of the recombinations could end up with all the little changes needed, Add a human / social situation of migrating humans and Boom. And the CFR may or may not be affected… and if it is it could go up or down.

Recombination scares the pants off me.

Curious,

Not much time right now to comment more than a few words . . .

Thank you for posting here.

The software that I’ve seen is designed primarily to exclude / identify recombinant sections versus randomly mutated sections for a very specific purpose.

Remember that a set of instructions that are designed to exclude will not necessarily have a rich heuristics engine and will likely have a far smaller content processing database than a software designed to include. These programs were designed using human-introduced factors and parameters that in the future will likely become much more labile as we learn more about how recombination occurs.

Many of the tests use a mutation rate factor per viral species per viral gene segment per host species, in other words, an average or a guess, to establish the potential for either accumulative random mutation or recombination in a particular section of a gene segment. Each of here knows that these types of factors are fallible, very fallible in situations that press for high adaptation.

I have seen no software yet that is based on the recognition of a rules-basis recombination. The current packages use foundational assumptions that disqualify them from viability to the questions being posed here. A software package that is built based on rules-based recombination would back-track identity for identity into potential ancestry based on geography, species, likely zoonoses, modes of transfer, et al. This back-tracking could be user-adjustable from sharp (identity) with few heuristics against a large database to fuzzy (partial homology) with massive heuristics against a potentially smaller database. As you can see, with identity being the driver, the software instruction set would be rather miniscule, relating more to parallel database access and processing than to neural logic.

I wait for this software to be developed.

ducksoup – at 10:32

I appreciate your analyses and can see that you are working from term to term and seeing some of the inconsistencies as well.

Feel free to write me at my address in my NS1 Profile.

The precepts that I hold on these topics are somewhat novel.

Goju – at 22:29

No. If we aren’t looking for rules-based recombination, we’ll call it a random mutation and dismiss it as unimportant in most cases, especially a SNP.

No. Changes frequently occur in all sizes.

Yes

Small changes are quite consistent with our current findings. Either way works. Like Frost said . . . Some say the world with end with fire . . .

NS1 – at 06:01

‘’Anon-YYZ,

Will you allow Niman free usage of your coinage, Niman-Inside, if Recombinomics does prove satisfactory?

I think its very clever. I still don’t understand the source of the powerful emotion behind your repeated, vindictive affronts? Do you believe that sarcasm makes casual personal attacks kinder or more stimulating to the discussion?’‘

As I read it, your first paragraph is sarcastic and in your second paragraph you are attacking my personality.

Vindictive, affronts are emotional nasty words. I didn’t and wouldn’t use them to project on any one. You may want to reflect on that.

Since you question my motive, I will ask you to re-read my post

ANON-YYZ – at 02:03 in which I said

Since you asked, I will tell you I am neither a scientist, nor work in the health care profession. I am not a PR executive, promoter, investor nor do I work in any field that puts me in a position of preferring one scientific theory over another as far as recombinomics is concerned. I am simply a prepper with a deep sense of wanting to know the truth. As many have said, the stakes are too high to be partisan. As a general stance, I am against sensationalism or excessive promotion.

I will add that I have no goods and services to peddle on this forum. There is no conflict of interest. I now have to wonder whether you have a conflict of interest.

I know why you are so upset and why you used those nasty words. In my posts, I exposed pseudo logic and falsehoods and de-bunked Recombinomics claims of being able to predict the future. Fortunately, we are not at a stage that we need to play Myth Busters – too many lives would have been lost unnecessarily if we had to go there.

This was not the first time you attacked my personality (previously in New Cluster in Egypt thread). I have chosen not to go down to your level. I am only a prepper. There is no need to discredit my character. Every one on the forum can judge. We have a difference in opinion and that’s just fine. We can debate the validity of Niman’s claims.

By attacking me personally, you are avoiding the real questions, as did ducksoup. My points are in my post:

ANON-YYZ – at 04:49

I waited many hours before I responded to your accusations. I didn’t want to interrupt Mary in Hawaii’s latest question, which was a repeat, and remains unanswered.

As for the Niman-Inside moniker, I stated clearly in my posts credit belongs to Intel Corp. It was really my most accurate way of describing how I see Niman’s marketing. You can go ask Intel whether you can use the brand name after you have a vaccine to market.

Anon-YYZ,

If you’d have done your homework, you’d see that I very openly confront the failures of the vaccine industry. I have no interest in any way in a vaccine for any disease, including H5N1. Neither I, nor anyone under my counsel, will be developing or accepting a vaccine for H5N1 should a pandemic occur.

Accusations without merit will leave you standing on only one leg the first time, unbalanced to be sure. After that you have to ask yourself, “Can I fit two feet in my mouth?” before you make any further accusations.

I do not require or wish an emotive debate with you, Anon-YYZ. I’ve followed your antics with amusement and now disdain as you’ve disrupted the exchange of information repeatedly.

Please save yourself further cortisol release and consider the facts before engaging (homework).

Anon-YYZ,

When I asked if you’d allow Niman your term, Niman-Inside, I was making a firm request. I think it’d be an exceptional and ironic fit.

Your further proclamation shows your failure to engage the topic. If you have a personality conflict with Niman or just everyone else, I understand. But if you want to discuss the topic at hand, you’ll need to review the answers that the responders give to your questions.

If you ask me a question and I refer you to data and then tomorrow you ask again, eventually I see that you are not interested in the solution and you are engaged to detract from the conversation.

No one has asked you or anyone else to disprove recombination . . . its a fact, observable and repeatable. The question is how it occurs and can we make mid- to long-term predictions. The OBVIOUS CLEAR DATA mantra that you mock shows the sequences to be considerably related in sub-segment strings in hundreds of places. How the sub-segment strings got there is another question. The answers are in the sequences. We just want to sutdy them and discuss them . . . in peace. You’ve been shown sequences. You have the same ability as everyone else to ask pointed questions after you’ve done the background work.

Until then, you continue to be off-topic and a distraction of monumental proportions to those looking for answers.

Mary in Hawaii – at 13:05

Certain areas do tend to recombine in smaller segments than others in particular strains. A larger database would allow us to hone the rules to a finer edge. Niman knows where to look for some of them.

Remember, we call them introns based on lack of research typically.

NS1 – at 02:32

I wrote ANON-YYZ – at 04:49

‘’It is always possible for Nimans theory to be right. It just needs to be proven by the proponent of the theory. Time and again, when asked specific question, the Niman supporters will turn the question around, repeat the OBVIOUS CLEAR DATA mantra, and ask the querier to prove recombinomics wrong. To me, that is a dead give away that the proponents of recombinomics know there are flaws in the theory, and cannot answer the questions.

If recombinomics is so right, there is no need to be evasive, there is no need to quote analogies, there is no need to call existing scientific theories by the name of dogma, or any other names. All it takes is to self publish a scientific paper on Nimans website, and have a forum there to allow questioning of the methodologies being used to establish the proof. All other scientists submit to that standard. Why should the rest of the world accept a lower standard of validation and accountability? ‘’

You avoided quoting the most important part of what I wrote and you wrote:

Your further proclamation shows your failure to engage the topic. If you have a personality conflict with Niman or just everyone else, I understand. But if you want to discuss the topic at hand, you’ll need to review the answers that the responders give to your questions.

You still haven’t addressed the main point, and you are again turning the question around, and attacking personality. Talking about Niman’s personality or my personality or personality conflict is simply trying to avoid talking about the main point (which you conveniently took out from your quote of my post).

If you ask me a question and I refer you to data and then tomorrow you ask again, eventually I see that you are not interested in the solution and you are engaged to detract from the conversation.

You haven’t addressed the main issue. You are talking hypothetical and makes it sound like you already answered that. You have been avoiding answering the real question, and offering fragments of examples of recombination as proof, and have been avoiding the rigor of generally accepted scientific cross-examination.

No one has asked you or anyone else to disprove recombination . . . its a fact, observable and repeatable.

When did I try to disprove recombination? I challenged Recombinomics theory, not recombination. Again, you are leveraging the general acceptance of recombination being possible to confuse the lay person Recombinomics is the truth. You are still avoiding the main issue, which I quoted at the start of this post.

You have no arguments. You attacked me personally. That didn’t work, then you tried to deflect, pulling wool over our eyes.

Until then, you continue to be off-topic and a distraction of monumental proportions to those looking for answers.

And now when you have no arguments, you call it off topic and try to cut off debate. What are you hiding?

Remember. The topic is Recombination At Vaccine Summit. Isn’t it OBVIOUS and CLEAR (you should understand your Niman language):

No one’s buying the Recombinomics theory to make a Niman-Inside vaccine. It’s vapor-ware, hot air, no substance. It’s propaganda.

Anon-YYZ,

It is not incumbent upon me or anyone here to teach an individual who does not desire to learn. Those who desire knowledge, seek it. When they find knowledge, they study it.

Please refrain from commenting on my posts until your background research allows you to make claims that are are relevant to the topic and are more than emotional rants.

When you post a comment that is relevant, I will answer.

Ns1 at 2:36 “Remember, we call them introns based on lack of research typically.”

I’m not sure what you mean, or if we are talking about the same thing here. I was referring to the sequence of nucleotides in an RNA molecule that is part of the primary transcript but not the functional RNA molecule (also called an intervening sequence)which is excised before translation of the code into a functional protein. Although this is generally something occuring in eukaryotic cells, certain large animal viruses such as the adenovirus have also been shown to do this, but I have no idea if influenza viruses also have introns. My understanding is that introns are flanked by similar or identical junction sequences, which is thought to allow exons to be assembled in different combinations or juxtapositions. This would seem also to make recombination at such points possible, in my opinion. But I really am just throwing the idea out there for others to chew on.

Mary in Hawaii, Flu has a segmented genome consisting of 8 gene segments containing 11 genes. The three segemnts with two genes use overlapping reading frames, The are no introns.

The slides from the meeting have clear recombination at multiple positiones throughput the gene. The most dramatic examples are in PA

http://www.recombinomics.com/phylo/Canadian_Swine_PA.html

There are 6 sequences with long stretches of identity with a 1977 sequence from Tennessee. Since the list H1 sequences are from 2003 or 2004, the 1977 sequence was copied in the gold colored regions for over 25 years without any change.

Therefore the “textbook” descriptions of influenza replication do not fit the data, because there were no mistakes in the sequences circulating more than 25 years later. The same type of identity with another 1977 sequence is present in PB2.

For PA one of the sequneces has regions that exactly match a 1931 sequence, again showing that the copying represented by the blue coded sequences in circulation were copied error free for over 70 years.

These sequences show that recombination happenes throughout the gene, and is not limited to certain positions within the genes.

As far as the importance of recombination and reassortment is concnerned, the Z genotype was defined in H5N1 in 2004. Since then the sequences have changed because of recombination. There are two major Clades. Clade 1 is in Southeast Asia and was used for the first pandemic vaccine. Most H5N1 detected now is Clade 2. Three are so different that seperate pandemic vaccines are being made (Qinghai in wild birds and all H5N1 countries west of China, Indonesian in humans in Indonesia, and Fujian, in human H5N1 in China.

All of the pandemic targets are consider Genotype Z, but that designation is virtually useless in vaccine targeting because the genotype Z has changed dramtically because of recombination and requires 4 separate vaccines.

That number will increase this season.

Hi Everyone,

Thank you for the welcome. NS1, I have a project that would benefit from using one of these recombination programs, but unfortunately have not sufficiently researched them to make any intelligent comments as to methodology and the robustness of the test. Thank you for your explanation though, I found it helpful. I also did a short search on the NCBI book section for viruses and recombination by copy choice and I came up with a figure and this explanation below. (I’m sorry the figure does not paste into the post), but the explanation of the figure below of how copy choice happens makes sense to me. I work on a retrovirus and by sequence analysis have found one variant that is co-expressed with another variant. The sequence of the one variant is exceptionally stable and has not changed in over thirty years of monitoring, while the sequence of the other variant is a clear recombinant. In developing quantitative PCR tests for the one variant which is extremely stable and has not changed we were forced to go to extreme conditions and even change the polymerase as one polymerase just could not seem to read through. Computer analysis of the secondary sructure of the extremely stable variant PCR product indicated a very tightly balled structure which only becomes uncoiled at extremely high temperatures. Now, this is in contrast to the clear recombinant which was easy to develop a quantitative PCR test for. Upon computer analysis, it had no such similar secondary structure. Thus in my simple mind, the explanation of areas of secondary struture inhibiting polymerase jumping makes sense to me. Could secondary struture prediction be applied to influenza to predict areas which may be more recombigenic than others? (Figure 43–4. Recombination by copy-choice of incompletely linked genes. The genetic interaction of certain RNA viruses can result in copy-choice recombination. In this mechanism, the polymerase begins replicating RNA template. By an unknown mechanism, which may involve a high degree of secondary structure in the viral RNA template, the polymerase complex (with its nascent viral RNA molecule) reassociates with the template viral RNA of different parental virus, such that the nascent RNA molecule will be synthesized as a novel recombinant virus whose RNA genome contains genes from each parental virus type.)

Curious

I can post pics if you email me at demfromct@earthlink.net. Or, if they are hosted, just copy the URL here.

You can use

http://allyoucanupload.webshots.com/

for pic hosting.

Upload the pic, then cpoy and paste.

Curious,

The 3D structure has come under discussion as to its speculative effect on rate of mutation, but I’m not sure if we have any data on the relationship to the measured rate of recombination in an area based on that area’s particular conformation.

Perhaps someone with an in-depth understanding of Niman’s rules will comment about the possible correlation between Niman’s landing zones and your clear example using the high degree of secondary structure. I have my own loosely formed theories about attractant areas with certain magnetisms (for lack of a better description) that I’ve yet to spend time researching and proving. My concept of magnetism / tendency to jump in particular areas may align with what you’ve noticed.

Who are some of your research subjects? Old stuff or new stuff?

Curious,

Please drop a few more examples in here of what you’ve found on the quasi-species model in your current research?

Is the recombinant varied consistently along certain residues or is it varied throughout the string? Have you published at genbank? Could we get a look at the stable sequence and ten or so of the recombinants (with conditions)? Feel free to send the sequences or a ClustalW alignment input to the address in my NS1 Profile. More info is better.

Away for a few hours.

Note copyright: this is for teaching purposes and is fair use

RBD at 08.57, or anyone else who likes to comment: bear with me, I’m just trying to learn this stuff… but hopefully I’m not the only one who’s confused. You write “Therefore the “textbook” descriptions of influenza replication do not fit the data, because there were no mistakes in the sequences circulating more than 25 years later.” Can I try paraphrasing your argument to see if I’ve understood it and then ask some followups? I think you’re saying:

begin paraphrase:

Conventional wisdom is that flu viruses evolve mostly by random mutation: that is, “mistakes” in replication. Given the extent to which flu viruses change from year to year, in order for this to be true they have to have a very high rate of random mutation: that is, the chance that any individual nucleobase gets changed during a replication has to be very high (compared with something-or-other). However, if that were the case, we would expect to see changes throughout the flu virus genome, and in fact we don’t: we see in the sequences that long stretches are not changed at all. So the mutation rate must actually be much lower. That being so, we need another explanation for the year-on-year changes we do see in flu viruses, and the only one that fits the bill is recombination. Therefore, long stretches of identity over decades in the genetic sequence of flu viruses is evidence that recombination is important in them.

end paraphrase.

In other words, it’s not that *this particular* pair of identical sequences proves that recombination must have taken place; that’s why you see talk about the work on different methodologies for identifying particular recombinations as irrelevant. Your argument is, if I understand correctly, that the presence of so many long pairs of identical sequences, regardless of the details of what they are, proves that recombination is an important mechanism. Is it? If I’m misunderstanding, please expand the argument yourself.

That argument seems to make sense, provided that:

1) we know enough about “random mutation” to know that it’s reasonable to expect it to happen at the same rate along the whole genome. Do we? On the face of it, it’s not obvious: maybe some parts of the genome are “harder” to copy correctly than others, maybe for structural reasons. Can that be ruled out?

2) we know that what we get is what we see: in other words, the fact that we see two sequences widely separated in time having identical segments really shows that there were no copying mistakes in that time. An alternative would be that all the possible mistakes are non-viable for some reason and get selected out. Can that be ruled out? (E.g. by degenerate codons? I’m confused about what sequencing actually shows.)

Thanks for any help.

Mary in Hawaii – at 04:59

I haven’t identified an area of the Influenza genome that is immune to recombination. RBD may have additional info.

hmm, the front page shows DemFromCT at 10.24 as the last poster on this thread, but when I refresh it, I see nothing after NS1 at 10.23. What happens if I try to post this?

I edited the picture and added the fair use statement. ;-)

That’s why you didn’t see a post from me. I’m one of the mods, and pogge, anon_22 and i may at times need to remove spam, edit format (not your words), etc.

Hi NS1,

You Write: (Perhaps someone with an in-depth understanding of Niman’s rules will comment about the possible correlation between Niman’s landing zones and your clear example using the high degree of secondary structure. I have my own loosely formed theories about attractant areas with certain magnetisms (for lack of a better description) that I’ve yet to spend time researching and proving. My concept of magnetism / tendency to jump in particular areas may align with what you’ve noticed.

Who are some of your research subjects? Old stuff or new stuff?

I will take the discussion off in a private e-mail because I don’t think the virus I work on is really relevant to influenza, but to answer your question about my model, I work on a retrovirus that causes mammary tumors in mice. Its an old virus, and there are many variants depending on the strain of mouse. There have recently been published some examples of recombination between exogenous and endogenous viruses based on sequence alignment alone. The example that comes to my mind is a recombination of an exogenous with an endogenous virus in a stretch of the LTR. The exogenous seemed to recombine several times with an endogenous that was co-expressed, mostly just short stretches of a few nucleotides. To my knowledge however, no one has ever used any of these recombination programs to study the recombination ability from this virus in a formal manner.

Thanks,

Curious

Curious, As I recall, sequence was used to demonstrate recombination in FeLV also.

Related to your comments on structure, most of the flu changes are third base transitions, so they are silent, but do substitute a purine for a purine, or a pyrimadine for a pyrimadine.

The substitutions for human PB1 are listed here

http://www.recombinomics.com/phylo/Canadian_Swine_PB1.html

All 16 position changes are exclusively transitions (and most are third base).

LifelongLearner,

You translation is generally accurate. What is not well appreciated by non-scientists is the fact that because of the redundancy in the genetic code, the third position in a given codon (which determines the amino acid determined by the DNA/RNA) is redundant, so it can be changed without changing the protein. These “silent mutations” would accumulate over time, if the copying was error prone (which is the basis for seasonal changes according to the conventional wisdom). However, the sequences for the swine have stretches that cover 1/4 to almost all of the gene with no such changes even though one sequence is from flu isolated in 1977, while the other is in 2003/2004. Thus, there were no third base chnages that mede it into circulation.

Curious,

Do the solo LTRs appear to have been integrated in the distant past or do you see signals indicating multiple integration locations with additions made during your test phase? Are LTRs consistent across tissue types? Is the inclusion labile to your testing or are you only seeing expression of existing LTRs and recombination with your test retrovirus? How long are the longest identities from the exogenous and endogenous? Do they consistently occur in the same locations? Could your retrovirus strain be transposing from within its own string independently (short strings?)?

I’m not sure if the software packages available are factored for endo/exo retro evaluation? Perhaps another contributor will have more certainty?

RBD: thanks. So that does away with my query (2) - since the sequences always seem to be quoted as sequences of amino acids (e.g. GER), and since I know nothing about how sequencing is done, I wasn’t quite sure whether the underlying sequence of bases was available for examination or not. If it is and it’s stable, OK. What’s known about (1), if it isn’t an impossible question? (Is there good reason to think that random mutation always happens at the same rate across the whole genome?)

LLL,

We definintely see differing rates of polymorphism across the various influenza gene segments.

LLL, The slides presented at the Vaccine meeting were all based on nucleotide sequence. so the identity includes all psoitions, including all of the third base positions which can be changed. The data indicate that stable “mutations” in flu are VERY rare (accumlating over decades or centuries) and are not the basis of seasonal variation), which is why recombination is considered “elegant evolution”. and represents a paradigm shift.

http://www.recombinomics.com/paradigm_shift.html

I’m back and will try not to get snipped again.

I have read through the thread and studied the charts, and I now understand, at least on a rudimentary level, what mutation, reassortment, recombination and quasispecies are. Or, shall we say, a little better than I did yesterday! One thing is obvious from this thread, and that is that there is a lot to learn on this subject.

I understand the point being made with the Swine PA chart, although I don’t know yet what PA is. I am so far at a complete and utter loss to understand the chart on PB1 Haplotyes.

I am afraid that in trying to absorb all the information in this thread, I have lost track of who has asked what, which answers have already been given, which questions have not been answered, or even if anyone is still interested.

At any rate, even if there is no further interest, I will continue to learn.

One thing is obvious even to me. The paradigm shift is inevitable.

NS1 – at 16:39 wrote “I’m not sure if the software packages available are factored for endo/exo retro evaluation? Perhaps another contributor will have more certainty?”

Shall I assume you have decided that would prefer to find another contributor? Because I don’t yet know what an end/exo retro evaluation is at this point. I am still back at the polymerase complex.

RBD at 16:21. While it is accurate to say that the third position is silent in regard to protein coding, it is very much misleading to say the third position is non critical!

There are several reasons for this. The three dimensional change induced by a diferrent nucleotide is enough to cause a critical reproductive failure in the RNA virus.

In addition, the packaging of the viral products is very much dependent on RNA sequence, and thus, changing any nucleotide may be enough to cause a viral failure. We do not know for sure, but it may well be necessary for a tit for tat mutation paradigm. ie in order to have one nucleotide change, another “package” dependent nucleotide must also change. This could indeed be a reason why we have not seen H5N1 break out yet. It may not be enough to have one or two changes to become transmissable. It may also mean that those “transmissable” changes have to occur simultaneously with another mutation that allow the lethal changes to not “knock out” the virus’ ability to package itself.

RBD at 16:21 …since I am merely a public school science teacher (best described as a generalist) and not a scientist per se (ie, no area of specialty or particular expertise) forgive any questions or ideas posed which are inane. However, that said, I have a question regarding this statement you made at 16:21

“What is not well appreciated by non-scientists is the fact that because of the redundancy in the genetic code, the third position in a given codon (which determines the amino acid determined by the DNA/RNA) is redundant, so it can be changed without changing the protein.”

Now, I have sitting on my lap my old college cell biology book, opened to the page on the genetic code which shows a chart of all the different possible codons and what amino acids they code for. An example: UUU and UUC both code for phenylalanine; however a change in the third nucleotide in that codon (which is what I interpret you to mean by ‘third position in a given codon’) to UUA or UUG is now a code for the amino acid leucine. There are several other examples of this as well. Isoleucine becomes a code for Methionine with a change of the third nucleotide to G, the code for tryptophan becomes “stop” with a change in the third codon, etc etc. Since a change in the third nucleotide of a codon apparently can and does change the amino acid in a number of instances, and a different amino acid would surely cause changes in the protein it is a part of, it would seem that I must be misinterpreting your meaning in that statement. Could you please clarify? Thanks.

working on it…

Mary in Hawaii – at 19:31

I see the ambiguity. Well, for example, the amino acid Phenylalanine can be represented by either UUU or UUC. As you know, both uracil and cytosine comprise the pyrimidine base. Therein lies the redundancy.

So, for example, both UUU and UUC represent the amino acid Phenylalanine, if the nitrogenous Nucleotide base is purine, represented by either A or G, well then the amino acid becomes Leucine.

I hope that clarifies, please let me know if I can further clarify (for us both!)

Mary in Hawaii,

So, in other words, a change in the third nucleotide may or may not changed the amino acid, however a change in the amino acid would, I believe, cause a change in the protein.

I hope this is clearer; I really should eat; brain is fuzzy from low blood sugar!

Mary in Hawaii,

By the way, to further clarify, all amino acids are comprised of at least two redundant codons. Some have 3, 4 or more, up to 6 possible codons, tri-nucleotide units. Amino acid sequences make up proteins. Therefore, if an amino acid changes, the protein changes.

Mary in Hawaii, The past three RBD (20:12, 20:52, 21:57) post are by someone posting nonsense, possibly in an attempt to destroy the thread.

In answer to your question, third base transitions involve replacing a purine with a purine or a pyrimadine with a pyrimadine. The examples you gave are transversions, which involve changing a purine with a pyrimadine or a pyrimadine with a purine.

It would be useful if the RBD, who posts really make no sense, would use another handle. There is no ambiguity, because transitions and transversion are precise terms.

pablo, I would never posts that changes are not critical. If such a statement was made by someone using RBD, it wasn’t me. Do you have a link to such a statement? Clearly someone else is posting nonsense on this thread using RBD.

RBD,

In other words, the third base codons that are likely to polymorph in recombination are silent typically because the nucleotide change will not result in an amino acid change.

Phenylalanine

• UUU
• UUC

A likely polymorph at the nucleotide level may substitute a C in the third position that was occupied by the U. The outcome is still Phenylalanine.

Whomever is posting as the fake RBD, please remove yourself quietly.

feel free to take the discussion to the new forum, where registration will prevent fake posters. in the meantime, we will investigate.

test

Gave someone a second chance and got played. I’ve edited the poster’s name on half a dozen posts. Playing games with the nickname and especially impersonating other members of the community is a bannable offence.

Good work, pogge.

Maybe I should make it clear: where it (at this point I take nothing for granted) posted as anonymous, I called it Banned. Where it impersonated RBD, I called it Banned as RBD. The real RBD is not banned.

suppose, evidence increases further that RBD is niman, would RBD remain unbanned ?

as 80% of observed differences are synonymous in 3rd position, then it’s obvious that these mutations are at least much more viable then ono-synonymous ones.

Or that these mutations just occur more often. But then most mutations should be viable - doesn’t make sense.

Hi NS1, you asked:

[Do the solo LTRs appear to have been integrated in the distant past or do you see signals indicating multiple integration locations with additions made during your test phase? Are LTRs consistent across tissue types? Is the inclusion labile to your testing or are you only seeing expression of existing LTRs and recombination with your test retrovirus? How long are the longest identities from the exogenous and endogenous? Do they consistently occur in the same locations? Could your retrovirus strain be transposing from within its own string independently (short strings?)?]

There are only a couple of published examples of recombination between an exogenous and endogenous retrovirus in my system that come to mind (there are several recent examples for HIV as well). They only looked at the LTR sequence as that is where recombination seems most frequent. In looking at a stretch of the LTR open reading frame of approximately 1000 base pairs, and aligning the sequence of the new recombined exogenous with several endogenous candidates known to be present in the strain there are usually stretches of just a few nucleotides (3–10)where recombination seemed to have obviously occured. Recombination had occured several times (5–10 if memory serves correctly) with the polymerase making several jumps from one template to the next. Sorry, this was not my work, and again I am not sure how relevant it is to influenza as this is a completely different virus. I should think wet work test tube experiments need to be done to test how often influenza actually does this. It may be possible to look for recombination if dual viral infection of MDCK cells were carried out. Careful thought as to the exact experimental conditions needed to see this effect would have to be given.

Sorry I can’t provide much more insight than this.

Curious

I’ve created a new Diary at the new site so that this important conversation can be continued http://www.newfluwiki2.com/showDiary.do?diaryId=264 Maybe individual posters can move some of the most important aspects of the conversation there.

I hope that you all - RBD, NS1, anon_22, Mary in Hawaii, disgruntled, ANON-XYZ, ducksoup, curious, LifelongLearner, et. al, - will migrate over to the new thread. The site does need registration, but it is easy enough to get an email address for that purpose at Yahoo or Hotmail.

Pixie – at 12:54

Can you please post a link in the new forum to reference this thread.

I think all of us who suffered from the impersonators will appreciate this thread moved over to the new forum.

Thank you.

The link is there! Pixie has put the link from there to here!!

it’s here

OK, the entire thread has been copied over for ease of discussion. Please move discussion to new forum, and I will close this thread to avoid duplication.