non-homologous recombination in cleavage site resulting in change in virulence
Random Mutation, Reassortment and Recombination
Part 1 - A disclaimer and a plea
Given the nature of this thead, there will some who will be tempted to see this as an attack on Dr. Niman. It is not. I have nothing but the highest respect for Dr. Niman’s many accomplishments. It was his site that first alerted me to the dangers of pandemic flu. His careful documentation of the H5N1 clusters was very important work. He has pushed for the release of influenza sequences for longer than anyone else that I am aware of. I agree with almost all of his criticisms of the WHO. As will become clear, my disagreement concerns his Theory of Recombinomics. My criticism will be directed towards this Theory, not Dr. Niman.
A plea - Please do not turn this thread into referendum on Dr. Niman. There are some people who like him and some who don’t. Please do not use this thread express either approval or disapproval of him (or me). Let’s try to stay focused on the science.
Finally, I am not a professional virologist. I have tried to be as accurate as I can, but it is entirely possible that I have made errors. I will be happy to correct them if anyone notices any.
Part 2 - Definitions
The nomenclature used to discuss flu evolution can be quite confusing. A number of times, I have noticed people cite papers that they thought supported their position but in reality supported a completely different position. This is an attempt to make some of the terms clear so that we may avoid confusion.
A reminder - influenza has a segemented genome. There are eight segments (genes) made of single stranded RNA. This RNA is composed of nucleotides. There are typically over a thousand nucleotides in each segment. Changes in the RNA can result in changes in viral proteins which may result in changes in the function of the virus.
Random mutation - fairly self-explanatory. May have a number of causes including inaccurate copying of genes by the polymerase complex. These changes usually are small, most often a single nucleotide. Such small changes are thought to result in Antigenic Drift if they result in small changes in the HA gene. This allows flu virsues to partially escape our immune system. This is the reason we need a new flu shot every year.
Reassortment - Exchange of entire genes between two flu viruses. This requires co-infection of a cell with 2 different viruses. Exchange of HA genes between different subtypes is thought to have been the cause of the last 2 pandemics. This large change is referred to as Antigenic Shift.
Recombination - This term actually has several meanings. In a general sense, it simply means the combination of nucleotides from different sources. Reassortment is thus one example of recombination. In fact, many papers with the term “recombination” in them are actually referring to reassortment. Further, genetic engineers use the term “recombinant” to refer to viruses that they have artificially constructed in the lab. This is actually the way that the term is most frequently used. However, for the purposes of this thread, I will use the term in the way that Dr. Niman uses it. That is, to refer to cases where parts of genes are exchanged creating a chimeric gene. For example, the first 700 nucleotides of an HA gene may be indentical to an HA gene from one virus while the remaining 1000 nucleotides may be identical to a HA gene from a different virus. As in reassortment, co-infection of two different strains or subtypes is required.
Recombination may be either homologous or non-homologous. The former occurs when the recombination occurs between the same gene or nucleotide segment. The latter occurs when two different genes are involved. Homologous recombination occurs during meiosis.
There is no doubt in my mind that all three mechansims of evolution occur. I do not know of any flu scientist who doubts that all three mechanisms occur. It is my understanding that Dr. Niman also agrees that all three mechanisms occur. The difference between the Theory of Recombinomics and Conventional Science relates to how often each of these mechanisms occurs and their relative importance in viral evolution *not* whether one of these mechanisms occurs.
Put simply: Conventional scientists believe that random mutation occurs frequently and recombination occurs infrequently while the Theory of Recombinomics suggests the exact opposite.
Part 3 - Recombination and Conventional Science
Some who post on flu boards have the impression that conventional scientists reject recombination as a mechanism for flu evolution. As I have indicated above, this is not true. In fact, recombination in flu viruses was first suggested by conventional scientists.
From Transfection-mediated recombination of influenza A virus (1992) Bergmann, Garcia-Sastre and Palese, J. Virology.
It should be noted that the frequency of these recombination events must be low since only a few recombinants have been identified during our studies and since we also suggest that the frequency of recombination is increased by our artificial transfection system. Nevertheless, our experiments suggest that the influenza virus polymerase does have the ability to jump between templates and/or to ligate different RNA fragments, and we assume that this property of the polymerase contributes to the observed evolutionary changes of influenza virsuses in nature.
So over 14 years ago conventional flu scientists demonstrated that recombination did occur in flu viruses. They felt this was a rare event then. They still think so today.
However, because an event is rare does not mean it is not important. After all, pandemics are rare. Some flu board posters have suggested that the Theory of Recombinomics has not been accepted because conventional scientists fear it will detract from the importance of Reassortment in generating pandemics. Yet, a paper in Science magazine suggested that there was:
Recombination in the hemagglutinin gene of the 1918 “Spanish flu”. This was published in 2001.
Science is one of the most prestigious journals. The same authors also had no trouble publishing a similar article in another journal:
The haemagglutinin gene, but not the neuraminidase gene, of ‘Spanish flu’ was a recombinant. Also published in 2001.
So, it would appear that Conventional Scientists have no trouble with the idea that Recombination occurs or that it can be very important in flu evolution.
I believe all 3 could be at work, with a lot of reassortment, some random mutation and perhaps a small smattering of recombination under unusual circumstances. What I don’t believe is that large sequences can remain completley static for decades. Perhaps a discussion of exactly HOW samples are collected and analyzed (i.e. how many virions are actually sequenced as the result of any one sample evaluated) would help to shed light on exactly how these sequences are derived. Having more sequence data to evaluate would be nice, but I won’t go there.
Part 4 - Problems with the Theory of Recombinomics
Now that we have established what Conventional Science and the Theory of Recombinomics agree on, that Recombination occurs and that it is potentially very important, we can now focus on where these two schools of thought diverge - The Rate of Mutation in flu viruses and the Frequency of Recombination.
The rate of mutation in flu virsues has been measured in a number of ways, including:
These studies all agree that the mutation rate of flu viruses is high. The papers cited above are a small subset of the total number of papers. There are over a thousand scientists who have participated studies measuring mutation rates in flu viruses. They all agree that the mutation rate is high for flu viruses. To my knowledge, the Theory of Recombinomics does not address this body of work in any way. A theory that fails to account for a large body of peer-reviewed work cannot really be considered a theory at all. It has failed at its inception.
What is the evidence that Homologous Recombination occurs frequently during viral evolution - none that I know of. The many experiments done to study the evolution of flu viruses have found that this is a rare event. In a paper that was published in March 2006 in Science the issue of the frequency of homologous recombination in flu virus evolution was specifically addressed. Here are the results:
Our findings suggest that homologous recombination is a rare occurrence in AIV evolution
From: Large-scale sequence analysis of avian influenza isolates
There is clear-cut evidence that recombination occurs. But this does not speak to the issue of the frequency of it’s occurence.
As near as I can tell, the only argument in favor of the Theory of Recombinomics is several examples of flu sequences that are identical across many years. The idea is that these few examples prove that flu viruses must have a very low rate of mutation. Therefore, the rapid rate of flu evolution must be explained by another mechanism - Recombination. This would then imply that Recombination occurs frequently.
The best evidence for identical sequences across time was recently retracted by the submitters. There are a few additional examples. I agree that these anomalies should be explained and not be brushed under the rug. I think they are potentially very important and should be investigated further. However, this is not sufficient evidence to disregard 70 years of work and hundreds of experiments documenting the high mutation rate of flu viruses, IMO.
I can think of a number of alternative explanations for these examples and will discuss some of them in a another thread.
Part 5 - Conclusions
I do not wish disuade people who wish to believe in the Theory of Recombinomics. People can believe in anything they wish. They should just be aware of the fact that there is a large body of data that flatly contradicts this Theory and that none of the established flu scientists give it any credence.
Recombination is not Dr. Niman’s discovery, but he is its champion in terms of H5N1 evolution. So, we do not REQUIRE him for a discussion of recombination, but it would be VALUABLE if he were here to promote his viewpoint. Is he able to post to the wiki now?
Many Cats, I asked DemFromCT when Dr. Niman would be able to post on FluWiki and my understanding was today. I waited to post this until today as I agree he should be able to respond, if he wishes.
thank you Monotreme
moeb, you’re welcome.
I think my one point got lost among monotreme’s mega posts: Perhaps a discussion of exactly HOW samples are collected and analyzed (i.e. how many virions are actually sequenced as the result of any one sample evaluated) would help to shed light on exactly how these sequences are derived. It might help to know in terms of what we see as a single snap-shot as opposed to the many changes which may be going on within a single infection.
By single infection, I mean a single patient with tens of thousands of cells simultaneously infected and the differences which may arise in viral progeny as a result, particularly if the person suffer from a dual infection with a second influenza virus.
Monotreme -
Thank you for compiling this body of work. It will go a long way toward promoting a civilized discourse on the relative merits of the competing theories.
Sarge: “Thank you for compiling this body of work. It will go a long way toward promoting a civilized discourse on the relative merits of the competing theories.”
Oh YEAH? You got any EVIDENCE to back that up, CHUUUMMMMP?
Now Racter, we are civilized here as all our posts show. No fireworks on this site…. :)
Minor point, but I believe that there are actually 10 genes associated with the influenza virus. Two of the eight segments have 2 genes each.
Many Cats, sampling bias is always an issue with influenza virsues since they are so prolific. What kind of bias you have to worry about depends on which Theory you subscribe to.
Some of the papers I cite, specifically address the issue. Is there some way you think sampling bias could cause someone to think that random mutation is more frequent than it really is or recombination is more frequent than it really is.
Birdman, you’re correct. It’s just easier and more understandable to laypeople to say gene rather than genomic segment. Even in the mammalian world, the definition of a gene is becoming problematical.
Thanks Sarge.
Racter, very funny :-)
Monotreme,
Thank you for your most excellent explanation. Have you found any explanation anywhere for the low rate of recombination in flu viruses? Or do you have any hypothesis about that?
My understanding is that recombination is generally rare with negative-sense RNA viruses. But I’m not sure I understand the mechanism. Can you explain what ‘negative-sense’ means in practice, and how that might affect the process of replication?
“Racter, very funny :-)”
Thank you, thank you, I’ll be here all week…
anon_22, thanks. There are number of factors that affect homologous recombination. First, there has to be something for the gene to recombine with. For a single stranded RNA virus, that means co-infection must occur. Statistically, what is the liklihood of this happening, ie, how often are you infected with two different flu viruses at the same time? No doubt that this occurs when many individuals are infected, but probably in a small minority of cases. This is one reason why I think the Karo clusters and others are clear-cut examples of random mutation rather than recombination. Of course, reassortment also requires co-infection.
Another variable that affects recombination is the degree of homology between the two genomic segments that are recombining. In general, for homologous recombination, the longer the extent of identity, the more likely recombination will occur. This has been studied in great detail in artificial mammalian systems, ie, gene targeting. Thus, the relatively short gene segments in flu viruses may contribute to infrequent homolgous recombination. I base this on my experience with mammalian systems, which may or may not apply to flu viruses.
Racter, where were you last Saturday night? We could have used you.
Monotreme,
“For a single stranded RNA virus, that means co-infection must occur.”
Wouldn’t that argument also apply to reassortment? Does reassortment occur more frequently than recombination, and if so, why?
If recombination happens rarely or is inconsequential, how would Niman’s accurate predictions be accounbted for?
anon_22 – at 13:28
Wouldn’t that argument also apply to reassortment?
Yes. I mentioned this in my response to your previous question.
Does reassortment occur more frequently than recombination, and if so, why?
Yes. As to why - it’s easier. Reassortment simply requires that entire segments be exchanged. Recombination requires a complex arrangement of proteins and nucleotides to form. Also, see my point about the length of the nucleotides influencing the rate of homologous recombination. This factor would not affect reassortment.
Monotreme,
OK. I needed to go through what you wrote word by word a couple of times to get it!
Thanks!
anonymous, I think recombination occurs rarely, I don’t think it’s inconsequential. It may be what’s needed to start a pandemic.
I’m not sure how many of Dr. Niman’s predictions have come true or what his access to sequences is. Some of his predictions are pretty hard to interpret, ie, the Spring Bride prediction. I do think he accurately predicted a specific polymorphism that appeared in a Turkish isolate. It’s possible that this was due to mutation followed by selection. However, I grant you that that was an impressive prediction.
Again, I don’t doubt that Recombination occurs. No-one does, that I am aware of. Perhaps Dr. Niman has identified a rule for predicting when recombination is more likely to occur. This would not conflict with conventional science.
anon_22, no problem. Homologous recombination is one of the most difficult subjects in all of Biology, IMO. When I look at the diagrams that illustrate all the intermediate steps, I get headaches. It is still not completely understood.
Monotreme,
“Homologous recombination is one of the most difficult subjects in all of Biology, IMO. When I look at the diagrams that illustrate all the intermediate steps, I get headaches.”
I’m sure what you will NEVER think of saying that just to make us feel better. :-)
Monotreme - at 13:13
In general, for homologous recombination, the longer the extent of identity, the more likely recombination will occur. This has been studied in great detail in artificial mammalian systems, ie, gene targeting. Thus, the relatively short gene segments in flu viruses may contribute to infrequent homolgous recombination.
Would that extent of identity have to be a longer section in and of itself or could it be longer in proportion to the entire gene to make recombination more likely?
anonymous – at 14:01
Would that extent of identity have to be a longer section in and of itself or could it be longer in proportion to the entire gene to make recombination more likely?
I think both are true. In artificial mammalian systems, the shorter the segment, the less likely recombination will occur, even if the segments match perfectly. The length of a typical flu gene would be considered extremely short. Given a certain length of a segment, the more identity, the more likely recombination will occur.
Dem,
This is a most fascinating, thank you for directing me here. I would tend to agree with much of what you stated. It is impossible to estimate the weight Niman-ish recombinomics given the data available. And you may very well be correct in your assertion that rare events do not necessarily mean that they do not have great or greater impact on outcome. But how really can be sure? Trying to determine more about underlying mechanisms with just sequencing as a tool is at best very very limited. So many assay variables like sample number, sampling manner and method of sequencing not to mention the variable kinetics of BF replication. The sequences, is the biopsy material from avian muscle or lung? It is not unusual for different strains of virus to infect to a lesser of greater degree, different tissues. Assuming co-competing infection by different viral strains, you have to wonder if the more successful viral strain ends up dominantly at your biopsy site. That would lead to very misleading sequencing information
To really look at something productive in the lab with this virus it would seem that elucidation of it’s RNA directed DNA polymerase kinetic activity is key. You touched upon this, and I agree.
Personally I might focus on reverse transcriptase. I cannot say for sure but because DNA polymerase is by far the most important enzymatic interaction when directed by single strand homo or hetero polymeric DNA. Inhibitors here might work to get a victim past the 7–10 day period before afferent immunity begins. Chemicals like azidothymidine might prove productive without knowing exact sequencing. By the way I might exploit this if I were infected with a higher path bird flu strain.
But getting back to the kinetics since it seems central to your argument. Trying to determine this from untagged sequencing samples is all but impossible, isn’t it? A lot is available on reverse transcriptase kinetics, and much of it pertinent to this discussion I’d reckon.
Naplespark – at 15:50
I’m not sure if your comments are directed to me or DemFromCT. I’m not sure I understand your comments or what they are in reference to. However, the papers I cite at 09:31 include a number of different types of analyses. The same sample biases would not apply to all of them, yet they all give the same result - a high mutation rate of flu viruses. Some things in science are at an early stage of development and are not certain - the mutation rate of flu viruses is *not* one of these.
Here is a paper from 1955:
Mutation and selection pressure during adaptation of influenza virus to mice
Although more precise estimates of the mutation rate and a better understanding of the evolution of influenza have occurred since then, the fundamental finding remains: influenza viruses mutate, even in culture where the biopsy sample biases do not obtain, and are subject to selective pressures.
Selecting for specific types of drug resistance in laboratory strains of flu virus is well-documented. This would not be possible if recombination were the primary driver of flu evolution.
Naplespark:
I wholeheartedly agree. Rocks in a streambed might be accurately tracked to their sources in certain outcroppings upstream, even in the absence of robust theories for the underlying physics. Every time I’ve asked for details, all I got was chirping crickets.
Thanks for the excellent explanation, not sure I’m much wiser but that’s my fault :-)
Two questions arise in my mind -
1) is it possible for a mutation to occur that would make recombination more likely ?
2) if recombination does occur what benefits are there to knowing? I’m sure Dr Niman has explained but I can’t get more than a few sentences into his posts before my brain locks up.
ukbird -
An understanding of the various mechanisms involved in viral genetic change might allow for the manufacturing of vaccines sufficiently soon to soften the pandemic blow. Otherwise, discussion around these theories are a very interesting intellectual excercise, but not much else, IMHO.
But if recombination is an exchange of elements between two viruses how can you predict which viruses will combine and which parts of them they’ll swap? And thus create a vaccine. It seems like a very complicated game of roulette.
ukbird -
I agree, very complicated. Perhaps Dr. Niman can enlighten us all.
Thanks, Monotreme — very helpful! Do you have any thoughts re possible explanations for the apparent contradiction between frequent random mutation and sequences that remain unchanged over many years?
Name, you’re welcome.
This apparent contraction between frequent random mutation and sequences that remain unchanged over many years has been observed before - in 1971.
Recent human influenza A (H1N1) viruses are closely related genetically to strains isolated in 1950
Officially, there was never an explanation. Unofficially, many virologists concluded that Chinese scientists had been experimenting with a laboratory strain of H1N1 that had been frozen from 1950, infected themselves with it, and then spread to friends and family, and then the rest of us got it.
Before any tinfoil comments start, let me point out that there were two laboratory infections with SARS, one in Taiwan and one in mainland China. In China, the virus apparently spread to people who had no direct contact with the laboratory strain. Disaster was narrowly averted.
SARS case raises safety concerns
SARS case in laboratory worker in Taiwan, China
SARS. China dumps CDC head, probes lab
Ill Chinese lab workers didn’t handle SARS virus
China’s disease control chief resigns for SARS lab infection
[http://www.medicalnewstoday.com/medicalnews.php?newsid=8471|SARS outbreak in China due to poor laboratory security]]
I do not know whether there have been lab escapees of H5N1 in China. Recent behaviour on the governments part there and strange retractions of data and attempted retractions of papers raises my level of concern. Another possibility I am researching is the idea that a poorly thought out live vaccine reverted back to an infectious version. I wonder how many pigs in China have been vaccinated for H1N1 and the nature of the vaccine. I also wonder whether they were housed in close proximity to chickens that had been vaccinated with a live H5N1 vaccine. These are just speculations at this point. As Dr. Niman would say, the story is in the sequence. I whole-heartedly agree with his demands that all H5N1 sequences, and H1N1 sequences be immediately deposited in GenBank. I also think the failure to collect samples from mammals wherever human cases have been observed amounts to unbelievable stupidity.
However, it should be pointed out that some of Dr. Niman’s examples come from US labs. I do not have an explanation for these. I fully agree with him that these examples of recombinants with accurate sequence across 30 years should be acknowledged by the people who published them. Where we disagree is his interpretation. If one accepts the tenets of Recombinomics, no further investigation is necessary. Everthing is explained. If you don’t, some people have some ‘splaining to do.
Monotreme. Thanks.
One of the questions that comes to mind is…is the level of knowledge adequate for parties to start to configure a hypothetical pandemic virus.
It seems to me that if I was in an office with a group of interested colleagues with the required background education…that is probably what we would be doing.
Monotreme, thanks very much for taking the time to write up this material, it’s being very helpful!
In a coincidence, while checking some influenza nomenclature usage in Fields Virology textbook (4th ed. 2001), I also came across reference to the Russian H1N1 virus which matched 1950 virus sequences. Here is a short snip from the text (p. 1550), which was written by Peter F. Wright and Robert G. Webster. It acknowledges in an obvious way, carry-over from “a frozen source”.
“A third explanation for the origin of pandemic viruses is that virus that caused an epiddemic many years previously remained hidden and unchanged in some place since that time. The appearance of Russian influenza (H1N1)…This virus appeared in Anshan, northern China, in May of 1977 and subsequently spread to the rest of the world; it is identical in all genes to the virus that caused a human influenza epidemic in 1950. Where was this virus for 27 years? The possible explanations include preservation in a frozen state, preservation in an animal reservoir, or retention in an integrated, as yet undetected form in the genetic material of a human or lower animal. The animal-reservoir option is unlikely, for the accumulation of mutations would have continued. There is no evidence for integration of influenza genetic material into the host genome, leaving the most likely explanation that in 1977, the H1N1 virus was reintroduced to humans from a frozen source.” (end quote)
What was not said - that the “frozen source” was probably a freezer. That is my interpretation, considering that release from permafrost is also not a likely explanation in this situation. At least now there is something in print from an expert source that acknowledges this type of sequence carry-over through a number of years.
Tom DVM – at 21:12
Much has been made about the difficulty of making a pandemic strain because we don’t know enough about pandemic strains evolve. Well, we do know alot more than we did. I also don’t think you necessarily need to have sequence endpoint in mind to get to produce a strain with the requiste properties.
beehiver,
Thanks for the quote. I think most virologists think the 1977 strain came out of freezer, although some did not. I suppose natural freezing would be possible in certain areas of the world, but this would seem to be longshot.
Monotreme:
My comment asking about how sampling and evaluation are performed, while not directly related to the mechanics of viral evolution, I believe is relevant because we are all taking “the sequences” which have been published and are trying to derive possible mechanisms of evolution based upon what changes we see in “the sequences”. My point being that if you have 5 blind people and they observe an elephant, if one examines the trunk, another a tusk, another an ear, another a foot and another the tail, each will have a very different perspective about what an elephant is. If we are sampling and growing up A virus to get “the sequence”, what happens to all the other possible virions with DIFFERENT sequences (derived from random mutation, recombination or reassortment) that we may be losing as we get “the sequence”? There are tens of thousands of cells manufacturing millions of virions. Most will be similar, but are they ALL the same? As you point out, certain variants can be selected for in the lab depending upon the conditions you impose. Are the techniques used to obtain “the sequence” selecting for one while missing others with critical changes (i.e “selecting for” the trunk of the elephant while missing the tusk of the elephant)? I do not know enough about the procedures to be able to answer the question. If that is a problem, then we may not even be looking at the right part of the elephant (if we are to believe the sequence data at all at this point). That is why I think it needs to be discussed, to ensure that we can be confident in “the sequences” at the end of the day. And even if we get that figured out, there may be something else that we are missing (for none of those blind men could have an appreciation for the concept of grey). Just trying to make sure we don’t miss something. Like the H5N1 elephant in the room.
Monotreme — Thanks so much for re-initiating a discussion on random mutation, reassortment and recombination [the three Rs, huh?].
I had pointed to a bunch of articles on recombination in viruses (not all negative sense viruses, I know) back in the Karo H5N1 mutations thread because I am trying to understand what viruses are up to — and what we know about what they’re up to — and how we know what we know about them. (Before I go on, let me say once again that I am agnostic about mutations vs. recombination being the prime mover in viral evolution — I just don’t know yet.) Anon_22 very kindly summarized the articles I had found. And then the discussion (as we all know), unfortunately, fell apart.
So, now I’d like to ask a couple of questions I had in mind when I first posted those articles….
Or, maybe it’s just one question: How do the scientists researching influenza A viruses (or any other viruses for that matter) know they’re looking at mutations as opposed to recombinations — or even something else for that matter? My concern is methodology. Unfortunately, I know and understand little about genetics, chemistry, microbiology, etc. — so I’m really in the deep end here.
In looking back over the several articles I posted — and at several that I’ve found since then — I notice that most of the studies seem to involve phylogenetic tree analysis or some other sort of indirect analysis of the changes in influenza viruses. What I’d like to see are some direct analyses of what’s going on with viruses — I want someone to get some viruses to reproduce in a controlled environment so we can see what they heck they’re doing. There doesn’t seem to be many such studies around (if there are, I haven’t been able to find them — please, anybody/everybody post them here if you know about them — thanks!).
The most recent one I found is (unfortunately, I don’t have access to the article — but I’d really appreciate a copy if someone does):
Comparison of the mutation rates of human influenza A and B viruses
Nobusawa E, Sato K.
Human influenza A viruses evolve more rapidly than influenza B viruses. To clarify the cause of this difference, we have evaluated the mutation rate of the nonstructural gene as revealed by the genetic diversity observed during the growth of individual plaques in MDCK cells. Six plaques were studied, representing two strains each of type A and B viruses. A total of 813,663 nucleotides were sequenced, giving rates of 2.0 x 10(−6) and 0.6 x 10(−6) mutations per site per infectious cycle, which, when extended to 1 year, agree well with the published annual evolutionary rates.
Sounds good. Other articles which look promising are:
Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1
-AND-
Heterogeneity of the mutation rates of influenza A viruses: isolation of mutator mutants
These studies sound great (in my opinion anyway) because they are direct observations of what’s going on. But, back to my question — how do these researchers know that the changes they see are due to random mutations and not something else? I mean — ok — to find out the mutation rate of a given virus, you (ideally) get some of those viruses to reproduce in a controlled environment so they can’t recombine with some other virus (which everyone acknowledges does happen sometimes). From what I understand, the mutation rate is calculated as “the amount of change which would occur within each cell burst” (Parvin, 1986).
Ok. But, don’t influenza virons reproduce in the scores (if not hundreds) within one cell before the cell burst? Given that, why couldn’t the viruses be mutating and recombining with each other before that happens — the end result being a lot of changes? And, so, the ‘mutation rate’ appears to be awfully high….
Just asking. Thanks.
Also, wasn’t a similar concern raised by Niman in that changes were not seen because the virions were being grown in an avian as opposed to a mammalian culture setting?
Monotreme = Just wanted to let you know I received your e-mail. I think I have become very tranparent. lol. gina
‘’’Maybe this is off the wall… But it is something I have been thinking about for a couple of months. It seems everyone agrees that recombination occurs, yet the question remains How often? And Why?’’’
‘’’Now this is just food for thought…. If you look at the environment in which a virus replicates, (a cell) why would you not also consider the environment in which this occurs? There are many unknowns still in the world of biology that we still do not understand. Using stem cells as an example…. Since both are basic structures. We know that stem cells respond to unknown mechanisms to guide their differentiation into whichever cell they become. These may be proteins, enzymes, (whatever). This environment leads the stem cell to make changes. Different environments occur with different cell types: i.e. lung cells, muscle cells, etc. Different environments would also occur within different organisms: i.e. chicken vs human. (or anything else). (This is probably different even different from person to person, also.)’’’
‘’’When the virus replicates, why would it not also adhere to these same “pressures” (or principles) similar to stem cells. (Not talking exact here, just the mechanism or theory). If viruses reacted to the environment in a predictable way, then it would be somewhat the same for each species, right. (Not talking about parts of the virus that make it unique; like H5N1) Therefore, it would not take long for a avian virus to “try” to adapt to a human, once it is exposed to the “human” environment” . A new environment would possibly cause the virus to change in any and every way possible to adapt… which means using whatever is available to assist in the adaptation. Recombination, re-assortment, random mutation? Recombination with an already human adapted virus would be a good choice. So would re-assortment. The environment may play a part in the method. Of course, if the environment partially dictates the structure of the virus, couldn’t random mutation be a driving factor? This might show as recombination, but actually could be a consistent response to the environment for that species. This would cause it to show in many viruses that met specific conditions.’’’
‘’’Also, would this initial flurry of recombination or mutation be more prevalent at the beginning of adaptation and then slow later? And preserve its structure? This would account for some of the long periods of time that elapsed with no change in parts of the sequences.’’’
Some excerpts from Stem Cell Info; just FYI…. It is a strech, I agree to liken a virus to a cell…. But it is a thought.
http://stemcells.nih.gov/info/scireport/
. . . <snip> Genes that regulate body patterning in embryonic development are well conserved throughout evolution among both vertebrates and invertebrates [19]. <snip>
Genetic and molecular biology techniques are extensively used to study how cells become specialized in the organism’s development. In doing so, researchers have identified genes and transcription factors (proteins found within cells that regulate a gene’s activity) that are unique in stem cells. Scientists use techniques such as polymerase chain reaction (PCR) to detect the presence of genes that are “active” and play a role guiding the specialization of a cell. This technique has is helpful to researchers to identify “genetic markers” that are characteristic of stem cells. For example, a gene marker called PDX-1 is specific for a transcription factor protein that initiates activation of the insulin gene. Researchers use this marker to identify cells that are able to develop islet cells in the pancreas. <snip>
If this is so far out in left field, please let me know… Just a thought anyway. Hopefully, coherent enough to follow.
What was not said - that the “frozen source” was probably a freezer.
----------------------------
maybe because they were doing the same freezing
and didn’t want to provoke a discussion about it ?
beehiver, very interesting what you brought up about virus in a frozen state and Monotreme’s speculation about labs etc.
This is not to dispel any of those ideas but let’s just take one more step back and ask ourselves do we really know the whole story about what happens to flu viruses in the natural state? Do we have no other possibility than the man-made, escape from lab route?
I am thinking of the 1918 virus as the perfect example of the ‘elephant in the living room - the worst pandemic in the history of mankind and we have NO idea where the virus came from.
So before we start hypothesizing with more esoteric theories, I think we need a lot more data (yes, back to data) as to what happens to flu viruses in general in the wild. Any flu virus, not just the pandemic ones. Even the notion of aquatic birds as reservoir is only a hypothesis built on the fact that all the known HA and NA combinations are found in aquatic birds. The logic appears to be slightly simplistic if not flawed in that we see the different HA and NA genome in aquatic birds, therefore we conclude that that is where they all come from?
Going back to the 1918 virus, we now know that it is different from any subtype that has ever been sequenced, ie it does not seem to have arisen from anything that we know about. Taubenberger thinks that this indicates it came from a source that was not in circulation until shortly before the pandemic, and gives 2 possible hypotheses: it had existed in a form that was somehow isolated from general circulation (laboratories could be an example, although unlikely for 1918 given scientists did not even know about viruses then, or bodies being uncovered from the tundra?), or it might have existed/mutated in a species of wildlife that we currently do not know about (eg marine mammal?).
The only way we can answer these questions is to gather a lot more information than we currently have. That’s why the large scale sequencing projects to create a database of influenza genome is so important.
Many Cats: The “elephant” problem is true of almost any scientific problem. No one experimental protocol will be free of all possible biases. The reason why I am so confident that random mutation is frequent in influenza viruses is because many different types of protocols have been used, each with different types of biases. Yet, the result is always the same: high mutation rate. This has been verified over 70 years of research in hundreds of labs. You just don’t get much better than this for proof.
Theresa42,
“How do the scientists researching influenza A viruses (or any other viruses for that matter) know they’re looking at mutations as opposed to recombinations — or even something else for that matter?”
I would let someone else give the proper answer to this question, but when I wrote up this thread Influenza Genome Project I found this chart of 207 sequences a very good visual aid that might help a little in ‘seeing’ mutations or reassortments etc.
Theresa, the kind of controlled experiments you want have been done, many, many times. Please check my post at at 09:31 under in vitro experiments. I have more, if you want them.
Hurricane Alley RN, I hope what I did was OK. If you want me to remove anything, just let me know.
anon_22 – at 22:17 — Thanks. I’ll have a look at it.
anon_22, I don’t disagree about it being helpful to understand what happened in 1918. People did not know what a virus was back then, so it certainly wasn’t a lab escapee.
The reason I prompted to think lab escapee (or live virus vaccine gone wrong) is because of the sequences. I find it strange that the people who have produced sequences identical to sequences from 30 years ago have no explanation for this result. I can assure they do not believe in Recombinomics. If the virus is “burning” in any animal, from seals to cats, it’s going to mutate. If it doesn’t, someone has to explain why.
Monotreme – at 22:19 — Theresa, the kind of controlled experiments you want have been done, many, many times. Please check my post at at 09:31 under in vitro experiments. I have more, if you want them.
Thanks, Monotreme. The first study you have listed under ‘in vitro’ studies in your 09:31 post doesn’t appear to be an in vitro study. The other two I have seen — mentioned them in my post as being promising.
I’d love the references to the other studies you’ve seen if you get the chance, thanks.
Still — doesn’t answer my question as to how the researchers involved can be sure they’re looking at mutations and not something else. Any thoughts on that?
Off to look at the link Anon_22 provided…. Thanks guys.
Monotreme,
But quite apart from the few examples of the sequences being exactly the same, do you have any other reason to suspect the lab escape theory to be true?
Also, it could be that this is 3am and I’m not thinking right, but are you suggesting lab escape as a major source of our current H5N1 outbreaks? Or is that just a side issue that you are investigating just to explain those identical sequences?
Monotreme. Are you saying that China has been developing and using live H5N1 vaccines or was it a live vaccine only due to uncontrolled production methods?
anon_22 — …chart of 207 sequences a very good visual aid that might help a little in ‘seeing’ mutations or reassortments etc….
That’s a really cool chart, anon_22! Thanks for sharing it. I’m having a look at the related thread, too, and will probably have more questions after I digest it properly, but the first question that pops into my head is…
The chart illustrates changes in amino acids in these H3N2 isolates. Don’t we (or I) need to be looking at a chart like this but of codons or neucleotides if we’re (I’m) going to try to track mutations/reassortments/recombinations/whatever? I mean, there’s — what? — 64 possible codon combinations but only 20 amino acids, right? This chart — even though it’s really cool — doesn’t have a fine enough ‘resolution’ (for lack of a better word) for what I’m after. Know what I mean?
Hi Racter,
My feeling exactly, underlying physics especially on a molecular level is very important indeed since it is the errors in transcription that leads to mutations, and even perhaps recombination. Reading sequencing data alone is hard to interpret. First and foremost there are problems inherently incorporated in sampling technique. Different ones give different results. When even a slight bias means a great deal, well, you must be careful what you assume is important. Unless standardized, we may be seeing quite different results. Nasal swabs from live patients are quite different from biopsy material derived from lung tissue dug from burial sites. There are inherent differences, yet rarely does sequence data here cite methodology. So it is suspect, and though interesting reading not something I would rely upon.
Also, studies cited from 1955, well. They held weight in 1950 and shortly there after but frankly, very little useful information can now be derived from them. Biology has progressed since then in ways only imagined. Much is currently written in the journals about Influenza genetics, reproduction and dissemination. So why cite ancient studies?
I am just getting my bearings here and trying to determine if it will be a daily stop during my morning coffee (smile), so you have to excuse me if I seem a bit abrupt. It is certainly not my manner, but I am just a bit confused by the mix of people here.
Monotreme. How do you vaccinate a billion birds in a day? Use a modified live vaccine…of course they would be dumb enough to think about live vaccines…inject one bird, put it in a flock and the flock vaccinates itself naturally due to infection…talk about Russian roulette.
I think we have a new term Chinese Roulette.
Hi Tom DVM,
Actually, that is not so far fetched, creating a firebreak is, well, something to seriously consider here. And a billion birds would not need to be immunized, rather just the wild migratory flock.
Naplespark. I wonder if that is the reason we got into this mess in the first place.
Is this practical?
Oh I am sure that is a mess that can certainly happen but as yet, with a few exceptions like in England, has not occured. Labs around the world have lots of nasty viral stock which if released would be catastrophic. But H5 seems to be following a natural progression, diseminating via migratory patterns, mutating predictably. The real question is if it can outrun its own immunity. Can it find a way to adapt before all the wild birds, or most are immune. When a virus becomes endemic usually infection rates fall because general population infection has taken place. This virus will soon be endemic.
Hi Melanie,
Practical is a good question, personally I feel it is not presently. That said, it is worth exploration. The question is if H5N1 as it appears in the wild bird migratory flock is more dangerous than an earlier strain, one perhpas frozen in 1977 (smile) during random routine post mortem pathology? If you then release the 77 strain before migrations circumnavigate the world and return to their starting point, most if not all birds there would be immune. There are a lot of assumptions, the first being that there is immunity emparted by the earlier strain which can protect against the more evolved threat. Also, perhpas you might be giving it the reservoir it needs? Bio firebrakes are meant to interfere with the lifecycle of an organism. Did you ever wonder why flu seasons differ? It is mostly due to infection patters caused by weather and travel. And area critical to mass infection comes down with flu early, and prevents that area from being used as a hub. It is the reason early infections of major metropoplitan areas sometimes lead to milder flu seasons. So why not use the same fundamentals to slow progression of this virus before it reaches the human population? Just a thought…
Theresa,
Here you go:
Comparison of the mutation rates of human influenza A and B viruses.
Human influenza A viruses evolve more rapidly than influenza B viruses. To clarify the cause of this difference, we have evaluated the mutation rate of the nonstructural gene as revealed by the genetic diversity observed during the growth of individual plaques in MDCK cells. Six plaques were studied, representing two strains each of type A and B viruses. A total of 813,663 nucleotides were sequenced, giving rates of 2.0 x 10(−6) and 0.6 x 10(−6) mutations per site per infectious cycle, which, when extended to 1 year, agree well with the published annual evolutionary rates.
Evaluation of several independent clones. We measured the frequencies of amantadine-resistant mutants of A/Mallard/New York/6750/78 (H2N2) (Table 1) and A/Swine/Germany/2/81 (H1N1) (Table 2) to indirectly assess the mutation rates of the two parent strains. Because the variability of mutant frequency is inherently high, we evaluated multiple parallel, independently grown viral stocks. Extensive plaque purification ensured that all amantadine-resistant variants occurred during growth in the plaque or allantoic fluid and did not exist prior to culture. A primary resistant plaque would grow to become a viral stock that was comprised almost entirely of resistant viruses and therefore would be recognized immediately.
Mutant frequencies and mutation rates. As described in Materials and Methods, we performed plaque inhibition assays to determine the population size and the frequency of amantadine-resistant mutants, and then we calculated the average mutation rate for each strain and its SD (Tables 1 and 2). For A/Mallard/New York/6750/78, the mutation rate (mmall) is 3.2 × 10−5 base substitutions per site per replication and its SD (SDmall) is 4.5 × 10−5; for A/Swine/Germany/2/81, msw = 6.8 × 10−6 base substitutions per site per replication and SDsw = 1.5 × 10−5. Table 3 shows that our data correspond well with two values previously published for the mutation rate of influenza virus.
Tom DVM,
Check out this site. I’d be interested in your interpretaton.
I’m also doing some offline research with one of our Flu Wiki colleagues on this issue. Don’t know if they want to be named ;-)
Tom DVM – at 23:03
Or maybe just start spraying the live vaccine in the general vicinity of the birds. Of course, no mammals would dare get in the way of the vaccine.
I think that a “predicted” mutation happens because a particular area (Middle East) has an endemic infection of say H1N1 (not correct - an example) and when H5N1 comes to be in the neighborhood there are then lots of chances for the two to do some exchanges. I don’t see the significant accomplishment in this type of a prediction. I would think that lots and lots of both types of viruses would make it seem almost “predictable.” Am I missing everything as usual? (I am going back to work on our computers, now that is easy to understand…grin.)
Monotreme – at 00:34 — Thanks, Monotreme. The first article there I mentioned in my original post [21:44] — and I agree that sounds really interesting and promising (anybody have access to the whole article?).
I haven’t seen the second article — that also looks good. Seems to be another example of direct (as direct as it gets) observation of mutation rates. Many thanks!
I have to say though, I feel that we keep winding up back at my original question, though — I hope someone can clarify:
…how do these researchers know that the changes they see are due to random mutations and not something else? I mean — ok — to find out the mutation rate of a given virus, you (ideally) get some of those viruses to reproduce in a controlled environment so they can’t recombine with some other virus (which everyone acknowledges does happen sometimes). From what I understand, the mutation rate is calculated as “the amount of change which would occur within each cell burst” (Parvin, 1986).
Ok. But, don’t influenza virons reproduce in the scores (if not hundreds) within one cell before the cell burst? Given that, why couldn’t the viruses be mutating and recombining with each other before that happens — the end result being a lot of changes? And, so, the ‘mutation rate’ appears to be awfully high….
Theresa42, why couldn’t the viruses be mutating and recombining with each other before that happens
That’s a good point. But I think it has been addressed. If you look at my post at at 09:26, you’ll see I cite a paper that used an artificial system to study recombination in culture. The way they set up the parental strains, you could not miss recombination, if it occurred. It did occur, but at a low rate. This is the paper that definitively proved homologous recombination could occur in flu viruses, AFAIK.
Thanks for your insistence on nailing every last loophole. It helps me marshall the facts more effectively.
Naplespark:
Niman says:
“Homologous recombination happens when the polymerase is using one gene as a template. The newly created RNA then hops off the template 1 and lands on template 2 (the gene from the other virus in a dual infection).”
And if the templates are similar enough to result in a functional gene, bingo. So far, that’s not an earth-shaking revelation. Where things get special is where he says:
“Recombination follows specific rules that can be used to predict the sequence of emerging viruses.”
So the changes aren’t errors, exactly.
I haven’t been able to get whether it is the polymerase that’s special, or the sequences, or both. It’s extra special if it’s both, because the sequences not only have to be special themselves, but they also have to code for the special polymerase as well.
Monotreme – at 01:24 — Theresa42, why couldn’t the viruses be mutating and recombining with each other before that happens
That’s a good point. But I think it has been addressed. If you look at my post at at 09:26, you’ll see I cite a paper that used an artificial system to study recombination in culture. The way they set up the parental strains, you could not miss recombination, if it occurred. It did occur, but at a low rate.
Cool study. Yeah. I wanna see more like that!
But, I couldn’t figure out what the ‘low rate’ of recombination is from that study — could you? I mean, what the authors said was: “Although both of these experiments resulted in the isolation of novel recombinants, several other RNP transfection experiments involving different nonfunctional influenza virus RNA segments did not result in the rescue of viable virus. Thus, the frequency of this recombination event appears to be low.” (p. 7578)
“Several other RNP transfection experiments”? Several other? How many other?? “Appears to be low”? Low? What exactly do they mean by low??
They also seem to have conducted their study rather half-heartedly: “In the last two experiments, no attempts were made to rescue additional recombinant viruses.”
What “last two experiments” — and why not look for additional recombinants when that’s what the study was all about??
Monotreme – at 01:24 — One other funny thing from that article you posted at 09:26 — the researchers got a surprise when one of their viruses actually recombined with a sequence in the polymerase preparation rather than with the (intended) helper virus. How little we know about viruses:
“…the RNA was isolated by standard phenol-chloroform extraction (3) and compared by polyacrylamide gel electrophoresis (2.8% polyacrylamide-7.7 M urea gel) (3) with that of the helper virus influenza WSN-HK (Fig. 3). The NA gene of the helper virus (lane 2) has been replaced by a recombinant gene in the transfectant virus (lane 3). However, the rescued RNA was larger (lane 3) than the transfected RNA (lane 1). Direct RNA sequencing of the 5′-terminal 500 nt with appropriate primers revealed that the rescued segment contained the full-length transfected RNA plus an additional 124 nt derived from the 5′ end of the influenza A/PR/8/34 virus NA gene (Fig. 2). These additional influenza A/PR/8/34 virus-derived sequences contain a poly(U) stretch of 6 nt and can form a stem structure involving the 3′ end of the segment, thereby restoring a functional polyadenylation signal. Surprisingly, the additional 5′-terminal sequences in the rescued gene were derived from influenza A/PR/8/34 virus and not from the helper virus. Since the polymerase preparation was obtained from purified influenza A/PR/8/34 virus, we suggest that the additional sequences in the rescued gene derive from RNA in the polymerase preparation used for the RNP transfection. It should be noted that several additional bands can be seen in Fig. 3, lane 3. We believe that they represent defective interfering RNAs but no further analysis of the RNA preparation was performed.” (p. 7578)
Monotreme. Checked out your site on the Harbin Veterinary Research Institute. Not a lot of information. It seems it is more of an experimental farm then a teaching facility as they have only graduated 200 graduate students in forty years; not a large number.
I assume Harbin is in Guandong?
As those of us who spent too much time in university would understand…it takes only one ‘misguided god-like figure’ to create a heap of trouble.
I’m not sure if there was anything else to see.
Could you clear one thing up for me. I assumed all along that the vaccines being used in Asia were killed vaccines. Am I wrong on this?
Tom:
Harbin is up in the northeast corner, far from Guangdong (near Hong Kong in the south). see map and see map.
And as to how to vaccinate a billion birds in China? Everyone does one. Tongue-in-cheek, but meant to convey the vast size of China’s bird and human populations, which we don’t always grasp.
DemFromCt Thanks.
Theresa42 at 22:59,
It is true that that chart only shows amino acid sequences. However, in terms of tracking the evolution of viruses for the purpose of finding out how they behave, ie the macro rather than the micro picture, then those are much better in assisting our thinking.
Theresa42, I really wish we could get Dr. Palese to comment on this thread. His group did the work. You’re asking for interpretation of their definitions of low, which I cannot provide. Just bear in mind two things: they set up conditions in an artificial way to favor recombination and the authors did say recombination was infrequent. I have read many of Dr. Palese’s papers and I would be surprised if he endorsed the Theory of Recombinomics. My interpretation is that based on the many different types of experiments he has done, he thinks recombination is rare. To my knowledge, no-one has conducted any experiments that would suggest that recombination is frequent.
Please note, I think your probing questions are entirely appropriate. In science, it is expected that the interpretation of any experiment should be subjected to intense scrutiny. But if one suspects the conventional wisdom is wrong and that the interpretation of many different types of experiment are all wrong, it is incumbent on them to explain all existing data, not just pick and choose amongst the available data. All Theories are incomplete, but some have more explanatory power than others. We cannot choose a perfect Theory, we can only choose the one the explains the most data.
Note, I also think more experiments can and should be done to explore the role of recombination in flu evolution. As I stated on a number of occasions, I don’t think its inconsequential, I think it could be quite important. But the way to do this is to do experiments, write papers, submit grants, do experiments, write papers, etc. Given the work on this subject, I see no reason why conventional science cannot address the issue. I don’t think there is any blockade to any scientist pursuing this, if they think it’s important.
Tom DVM, more on Dr. Chen and vaccines later, on another thread.
Monotreme Thanks.
Hi Racter,
Actually, it is probably thermal. I worked with HIV reverse transcriptase for a bit and a lot of the synthetics demonstrated only partial transcription at higher temperatures. Assuming the natural model here fails in the same manner, perhaps that is what Niman is missing? Perhaps part of the RNA is transcribed, but released due the higher kinetic activity caused simply by higher temperature? If so it could allow second opportunities. I have read a lot of questions here pertaining to temperature of an infected host bird, so maybe it is. Birds do usually maintain take-off temperatures which seem quite high comparatively. Anyway, I am a big fan of kinetics, of exploring enzymatic activity at a molecular level. To wit, it is a good idea as part of examining the kinetic activity on a molecular level to view activity at differing temperatures and then try to model and predict what will happen.
Recently here I read a post in which a non scientist posed a question about temperature. As I recall the question was “If a person could be heated to kill BF.” It is an interesting question I thought. Yet the answer from one of the seeming scientists here was quite strange indeed. They said that it could, but the temperature would also kill the person. How strange, I remember thinking, when temperature regulation is often a weapon of first choice of weaponry for your body. Higher temperatures can lead to enzyme failures even when only slightly fluctuated. I might have answered that question saying that yes, it is a great thing to have your temperature elevated when infected with a virus since countless studies pertain to the altered kinetics aiding in everything from immune response to enzymatic failure of viral reproduction. Every little bit helps after all, and I am sure that is why we all evolved to carry the fever gene (smile). Some might even argue that taking aspirin to reduce your fever extends the length of infection when you have the flu. So I might have answered that question is a very different way. But that is the beauty of a forum such as this.
Naplespark – at 10:51
Moderation in all things. ;-) Human temp of 104 is different than 106 or <gasp> 108. best to define parameters (a normal temp is < 100.5).
Monotreme,
“As I stated on a number of occasions, I don’t think its inconsequential, I think it could be quite important.”
Well, there are two instances where recombination could have been important. Both has to do with the change from LPAI to HPAI of H7 viruses, in 2002 in Chile Recombination Resulting in Virulence Shift in Avian Influenza Outbreak, Chile and in 2004 in British Columbia Novel Avian Influenza H7N3 Strain Outbreak, British Columbia.
Both were instances of non-homologous recombination resulting in insertion of multiple basic amino acids in the HA cleavage site: 30 nucleotide insert from NP gene in Chile and 21 nucleotide insert from the M gene in British Columbia. Both happened in chickens and the resulting viruses were highly pathogenic to chickens.
Apart from these, there were 2 previous experimental situations involving multiple passages through chickens or chicken cells, also for H7, where non-homologous recombination resulted in a change in virulence from LPAI to HPAI. These are discussed and referenced in this paper which also summarizes the whole issue of non-homologous recombination resulting in conversion of LP to HP. Intersegmental recombination between the haemagglutinin and matrix genes was responsible for the emergence of a highly pathogenic H7N3 avian influenza virus in British Columbia
So, are these unique and unusual events? Or have we not looked hard enough?
Thanks to Jhetta for providing the references.
I would think that in the case of H5N1 it would be better to lower the temperature. Isn’t that what one of the modifications necessary for replication in humans rather than in the higher bird temperatures. I would think this virus wants to be in a host who has a fever….but computers look better and better all the time.
There is also the issue that in the case of someone in respiratory distress, higher body temperatures cause a significant increase in oxygen requirements, and this could make a big difference if they are in borderline respiratory failure, especially if you don’t have any oxygen or cpap available.
Monotreme – at 10:29 — Hey, thanks for your response, Monotreme. I agree with you — I think my probing questions are entirely appropriate, too. ;-) As are everyone else’s, of course.
I agree with you, also, that more research should be done on the role of recombination in influenza (and other) viruses — as should more research be done on the role of mutations and reassortment and everything else about viruses! Remember, I’m not a recombination advocate — I’m still just trying to understand.
Which brings me back to my original point/question (once again). Forget a moment about proving recombination one way or another….
In my first post in this thread (21:44) I raised the question how do researchers know that the changes that they see in influenza viruses are mutations (I’m assuming we’re talking about point mutations)? You pointed me to several articles about mutation rates in influenza viruses — and I found several myself — but none of them have answered my question (maybe the Nobusawa & Sato one does, but I don’t have access). Most of the articles you’ve referenced as well as the ones that I found deal with indirect observations — and even the few direct observation studies seem to make quite a few inferences about mutations and mutation rates in influenza viruses.
Now, that’s fine if scientists are inferring that what they see are, indeed, mutations and mutation rates — I can live with that. But, then, the whole certainty about mutation rates in all of the discussions around H5N1 is not so certain at all — another fact which I can live with. I just want to know for sure what the certainty about mutation rates is.
anon_22 – at 10:16 — Theresa42 at 22:59 — It is true that that chart only shows amino acid sequences. However, in terms of tracking the evolution of viruses for the purpose of finding out how they behave, ie the macro rather than the micro picture, then those are much better in assisting our thinking.
Thanks, anon_22. It is a very useful — and cool! — chart. And it obviously does help in understanding the evolution of viruses and how they behave [and maybe someday it will make some sense to me ;-) ]. I just don’t think it helps directly with answering my question about how one tracks mutations in viral genomes and how one calculates mutation rates in these. Clearly it helps with the bigger picture — it just doesn’t offer the detail I think we need here, though.
Theresa42,
I don’t know enough virology to even begin to tackle the question of how scientists arrive at the conclusion of high mutation rates. :-) However, if we ask it the other way round and say how does one recognize a recombination, and how is that different from the mutations, the this study that you posted originally in the Karo thread might give us non-specialists some clue as to how they might go about it. A Novel Approach to Detecting and Measuring Recombination: New Insights into Evolution in Viruses, Bacteria, and Mitochondria
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.’’ …etc”
In another paper, Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses there is a discussion of how the authors came to their conclusion that a change of the 1918 virus previously ascribed to recombination was actually more likely to be due to extensive rate variations in substitutions in HA1 and HA2. I am inferring from this the kind of methods that scientists might use to settle such questions.
“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 can only conclude that with many notable scientists working on these problems, and with peer-review etc, that they are able to come to certain conclusions about methodology. Now they might be all wrong, but as Monotreme pointed out earlier, if most of the scientists working on the same problem come to more or less similar conclusions, I am inclined to believe them rather than dissenters who have not been able to provide substantive research to back up their claims.
The bigger problem for me is that sometimes non-specialists may get sidetracked into trying to discern some difficult and esoteric aspects of science, and not enough attention is paid to all the lessons that we can learn from stuff that we do know about.
I am now a lot more interested in the nature of reassortment events which have been generating the various subtypes of H5N1 from the unique ecological environment of Southern China
H5N1 influenza: A protean pandemic threat
The evolution of H5N1 influenza viruses in ducks in southern China
My guess is if we have a pandemic from H5N1, this will be where it is going to come from, and not genetic drift from strains in Indonesia etc.
The above 2 studies were also discussed here
anon_22 – at 12:40 — The bigger problem for me is that sometimes non-specialists may get sidetracked into trying to discern some difficult and esoteric aspects of science, and not enough attention is paid to all the lessons that we can learn from stuff that we do know about.
I hope you’re not characterising my set of questions here as an attempt to “discern some difficult and esoteric aspect of science.” I agree, all of this is clearly difficult — and I am definitely a non-specialist. But what I am asking about is simply a clarification of the very basics about viruses — the fundamentals. Not something esoteric at all — at least in my view.
Forget about recombination. I’m not even interested in that right now. Let’s imagine this thread is entitled “How viruses work” or something like that.
Ok. We’ve been told that the main driver in influenza evolution is the acquisition random mutations. Great (I think Darwin’s ideas on evolution and the modern synthesis and all that are correct, so I’m not looking for anything more than a naturalistic explanation here.)
Ok. How do they know this? I’m looking for any and all direct, experimental evidence to show that what we’re seeing are mutations and that, therefore, one can calculate mutation rates, etc., etc. As we all know, reconstructions based on phylogenetic analysis are fraught with all sorts of problems — basically, your phylogenetic tree is just as good as the data you base it on — so I’m not (so) interested in such indirect evidence.
I’m just trying to think through this — not just believe everything I read, whatever the source.
Thanks again for taking the time to respond.
Theresa,
Have you looked at the Science section on the wiki? All of the information is there, and it takes up a little more space than an answer on the Forum could give you.
Theresa42
I wasn’t talking about you. Sorry if I gave the wrong impression. I just wanted to emphasize to lurkers and newbies that this is a very interesting discussion but it may not be the most important one. Apologies again!
Oh, I also wanted to share how recently I am getting more interested in reassortment and why. :-)
anon_22 – at 13:36 — I just wanted to emphasize to lurkers and newbies that this is a very interesting discussion but it may not be the most important one.
I would think that understanding the basics of how the virus works — and how scientists have come to that unerstanding — would be one of the most important discussions. What do we know — and how do we know that?
Naplespark -
Do you think it would be possible to directly observe recombination by radioactive tagging of a donor sequence? I could see problems with this as the isotope incorporated into the nucleotide decays (as it would break the molecule) - but if not radioactive tagging, how might the nucleotide be tagged?
unerstanding >> slang for understanding! :p
I have several rather stupid/basic questions, some of which I asked before on other threads and never saw a response to-
If the error rate/mutation rate in a virus is high, and if the numbers of identical sequence data across a span of years is low, how possible is it that this is simply a stochastic event? That is, apart from the “lab escape” hypothesis, is it possible that some of these sequences are simply statistical outliers? Has statistical analysis been done on these occurances? Do we have enough data for a meaningful attempt at such? Is the granularity of the sequencing data sufficiently fine to permit detailed examination of the exact ATCG combinations? I would be very interested to see whether or not the sequences showed any evidence of minor alterations that might suggest long term residence outside a “frozen environment”…..
Conversely, if recombination is the major mover for viral evolution/adaptation, how are the evident small shifts in flu virii, as seen by variation in infection rate and virulence in seasonal influenza and the 1918 pandemic, explicable other than by mutation?
What study has been carried out on the effect of environmental factors (temp, ph, various possible enzymes, etc.) on each of the “three Rs”?
I recall reading a book by Hoyle and somebody else whose name escapes me which asserted that virii may have evolved in comets, and that viral infections may come from cometary sources. It was an interesting idea; has there been development or debunking of this idea?
Finally, all branches of science are replete with examples of how conventional wisdom turned out to be wrong, and the only thing that changed the extant paradigm were just a few trivial outlying data points.
As an example, geologists have, at various times, rejected catastrophism and continental drift as major drivers in geological reshaping of the planet. Yet now, BOTH of these mechanisms are widely acknowledged to be operative. Is it possible that all three of the “three Rs” are major shapers of viral evolution, depending on local environment?
One last question:
Do we know which of the various genetic components in the flu virus confers immunity?
Trying to wrap my spinning head around all of these thought trains, the importance of context stands out for me. As LMW just noted “Is it possible that all three of the “three Rs” are major shapers of viral evolution, depending on local environment?” For example, could recombination be rare but relatively more important in driving unique changes important to shaping a pandemic?
The 2 studies cited above by Anon-22 show the HA genes drifting while the other genes have undergone reassortment (so it might not be either/or, but a combination of the two that does us in); other citations above show H7 went from LP to HP due to recombination (maybe it will involve all 3, or maybe specific mechanisms for specific changes, or maybe it depends on context?); Many Cats reminds us that seeing only the trunk might prompt a very different inference about what an elephant looks like.
So 1918 may offer important clues, especially if we can develop some useful theories of what the virus was up to during that mystery period between birds and humans. But even if we figure that out, it may only partly explain what’s happening now. Aspects of the current context—vaccines/lab practices, agricultural/industrial practices, even climate change?—could be offering unique opportunities, incentives—or, if we’re really lucky, last-minute barriers to a major pandemic.
The two things worrying me most at the moment (and they may be related) are: 1) What’s been driving not just H5N1, but other AI viruses to become more virulent in mammals since the 1980s (as noted in the Webster et al study that Anon-22 cited)? 2) The philogenetic chart Niman posted showing most human and bird isolates in Indonesia are not closely related, which raises echoes of that mystery gap pre-1918.
Hi Sarge,
Hmmmmm, the use of stable isotope to label and biomark RNA synthesis products for the purpose of selective sequence identification, well, sure why not? The degree of isotope enrichment is the key. And ultimately depends on the kinds of answers you wish to derive from the questions you are posing through your experimentation. Depending on what you need to know, problems would need to be overcome, shared precursor pools for example. Recombination in a Niman sense of the word, well, is a bit different, and presents it’s own host of special issues. Assuming magnetic resonance spectroscopy and some electrophoresis in combination, still it would be a fun problem solving to end up with clean unbiased data.
On a personal note, I have never gotten the hang of electrophoreses. I had a lab partner who once who for 6 years did it day after day flawlessly, and always got perfect results, but for me nada (smile).
LMWatBullRun:
Like virulence, immunity is not a property which belongs exclusively to either the virus or its host, but which emerges out of the complex interactions between the two. One of the things that makes recombination seem so heroic is that since some aspects of viral replication (such as the “capping” and “splicing” of nascent RNA) appear to depend heavily on cellular mechanisms, it is hard to accept that what worked in one host would work the same way in another, especially when the hosts belonged to different species.
Naplespark -
The sort of thing I was considering might involve the cell cultivation of one virus, where the cell culture is imbued with an isotope of say, carbon-14 in the nutrient medium. Long half-life seems to me a plus. Exotic cyclotron-derived isotopes of oxygen, etc. like for PET would seem too short-lived and thus too high in specific activity to last through viral amplification - plus I think the emitted energy and decay transmutation (I have no idea what the decay chain for PET isotopes is) would break the RNA.
The isotope is incorporated during the intracellular assembly of the virions. Another culture is made and co-infected with another strain. Maybe we get to see directly the exchange at the nucleotide level, ala’ Niman recombination? Ah, then again maybe not. We would be looking for the proverbial needle in a haystack - a single occurrence of a nucleotide substitution among zillions of replications. Probably wouldn’t even be detectable as a whole-gene reassortant. Oh well…
Hi Dem,
Yes I totally agree, 105 is for an adult, well, angelic (in a meet them sort of way), kidding of coarse, but humor is what makes life fun (smile). Seriously, even small fluctuations in core temperatures tend to have an effect on the real time expression of immunity. At the heart of this temperature change is kinetic activity.
As far as higher temperatures favoring BF, well, perhaps that is the case, perhaps the higher resting temperatures of birds is built into the activity of the genetic code of BF and is subsequently imparted to us when infected. Still, I trust human fever, not too high as you most elegantly point out, but high enough to contribute to the defeat of an invader in us.
Coarse, a fever may just be your body telling you to stop what your doing and don’t be a total fool!
Racter-
Sorry I did not state my qustion more clearly. I am not a virologist, but my parents were both immunologists, nad I have picked up some limited knowledge along the way. The workings of the immune system are indeed complex, no doubt, but there are some things that are known about some pathogens and what specific factors the human immune system recognizes are previously known invaders for those pathogens. I was wondering what the state of the art with regard to influenza was.
More specifically, which are the genetic components of influenza virus to which exposure in/infection of humans confers immunity? The fact that immunity appears to be at least partially lost in months/years suggests to me either that the flu virus IS undergoing constant small change, or that the immune response is more complex than for other viral infections, or that I know too little to have an opinion. <grin> Probably the third.
I really wish my late father were around to discuss this… sigh.
As it is, I eagerly await the response of the more learned on my other admittedly basic questions…..
Hi Sarge,
Very interesting indeed, my quick though is that I think more stable isotope probing might be more what you want to use. So think SIPs. And sssuming you can tell the two RNA’s and their re-assortment/recombination products apart, I would isolate a bird, use 2H2O, tag the donor for weeks, then co-infect it, and look at the RNA synthesis products using an NMR. If you can distinguish biotags from both strains, well, voila, it has to be recombination. And also the beauty is you can isolate the new combined virus from the urine of the animal, hard to do with birds I know. Oppps but Dr Niman has already patented that process for mamals anyway, so we would have to take him out to dinner for that privilege (smile).
I do share you concern about molecular bonds breaking given the increased kinetic energy of the isotopes you use, but all you have to do is verifiy, not quantify so your task is quite simple. Oh and also, I hate to work with canisters which have a biohazard label on them, makes me think I may glow later.
Sarge
Oh and weight matters too, isotopes are heavier, so, keep that in mind if you explore this.
LMWatBullRun:
Receptor affinities seem to get most of the attention. As much as anything, this is a matter of “looking where the light is best”. It still makes as much sense to look at this from the perspective of differences in the distribution of ciliated versus non-ciliated cells in various hosts, but it’s entirely possible that the humans so far infected are not unique in this respect — and that they yet have a genetic predisposition to H5N1 infection, just one that has nothing to do with cell surface receptors. A wordy way of saying “we don’t know”.
No one denies that influenza constantly changes. This fact alone guarantees that the immune response is more involved than for other viruses.
I wish my late father were around to discuss this too, and many other things as well. I should probably just count myself lucky that I’m still around to discuss things.
Naplespark -
I’m flattered, but I won’t be conducting this investigation. My lab experience is mostly from undergrad days, and during the Carter administration. I have some VERY limited experience in BSL-3, but that was strictly orientation. The truth is, I’m a dilettante.
But, I found your perspective interesting. I believe that investigations of the interactions at the molecular level, if such could be undertaken, might be the only way to put this argument to rest.
Thanks again!
Name – at 15:16
“The 2 studies cited above by Anon-22 show the HA genes drifting while the other genes have undergone reassortment (so it might not be either/or, but a combination of the two that does us in);”
Yes, I think so. The studies from Webster’s group would certainly suggest reassortment happening on top of whatever antigenic drift that’s going on.
“other citations above show H7 went from LP to HP due to recombination (maybe it will involve all 3, or maybe specific mechanisms for specific changes, or maybe it depends on context?);”
:-) That is exactly what I am wondering about too, whether those were unique and unusual occurrences or whether they are part of a pattern that we are failing to see. But I had searched for citations of these papers and it doesn’t seem to me that there was anybody else following up on this question or any other instances after 2004. If anybody finds any please let us know. The fact that they were all a) H7, b) occurred in chicken or chicken cells c) non-homologous recombinations involving the cleavage site of HA seems to me more than a coincidence, but I haven’t seen anything that might explain this, and whether it might apply to H5. I can certainly make a case for this being a possibly important mechanism for H7 to become a pandemic strain.
“So 1918 may offer important clues, especially if we can develop some useful theories of what the virus was up to during that mystery period between birds and humans. But even if we figure that out, it may only partly explain what’s happening now. Aspects of the current context—vaccines/lab practices, agricultural/industrial practices, even climate change?—could be offering unique opportunities, incentives—or, if we’re really lucky, last-minute barriers to a major pandemic.”
I certainly think that the fact that we have not solved the mystery of the origin of the 1918 virus is hugely significant. Taubenberger’s ongoing research at the NIH on pre-1918 would hopefully shed some light on this. He and john oxford are teeming up to sequence autopsy samples of suspected flu cases from 1905 −17 from the Royal London Hospital. Apparently they used to do autopsies on every death at the hospital, so there’s a huge archive sitting in basements with clinical notes and tissue samples etc. They’ve identified 200 patients who might have died from influenza plus 200 controls, and will do the painstaking process of trying to find fragments to piece together, like what they did with the 1918 virus. They hope to find what was in circulation prior to 1918, and compare those to the 1918 virus. This is going to take some time, but they have recently identified at least 3 samples as H3, which is exciting and slightly mind-boggling.
“The two things worrying me most at the moment (and they may be related) are: 1) What’s been driving not just H5N1, but other AI viruses to become more virulent in mammals since the 1980s (as noted in the Webster et al study that Anon-22 cited)? 2) The philogenetic chart Niman posted showing most human and bird isolates in Indonesia are not closely related, which raises echoes of that mystery gap pre-1918.”
The first one worries me too, because the combination of ongoing reassortment events driven by unknown mechanisms from unknown donor viruses and increasing virulence for mammals is very bad news.
The second one I think we need far more data to draw conclusions, because it is very hard to compare samples unless you have more epidemiological information eg did they take avian or pig samples from the same location as the human cases? Andrew Jeremijenko’s comments give an impression of very unsatisfactory surveillance in Indonesia, which is very unfortunate.
Hi Sarge,
I think you’re right on the money! Understanding the kinetics is the only real way to understand the mechanism. Usually shedding light in such areas is a slow process leading to indefinite conclusions, but I do not rule out Niman’s view. Rare occurrences in nature often are utilized in ways which are hard to understand. Most people, researchers included, often make this mistake. He may be on to something here.
Molecular kinetics though well studied, is itself not as well understood as a chemist might lead you to believe. Computer models really help here. Still though I often feel that the fudge factor, bias, drift, etc often occurs more as a result of kinetics than poor lab technique. Other may disagree, but those would be the ones that have never spent years in a lab (smile).
In nature seemingly random events often are utilized. The beauty is often found in the nuances, as I suspect is the case with Avian Influenza. If recombination occurs frequently enough to direct evolution when other mechanisms fail, it surely will prove to be rare but important.
I think Niman enjoys writing patent applications, and his thoughts perhaps are more targeted towards a useful purpose for his ideas. To be ahead of the curve you have to over assume, a mistake for most of us, but a great benefit to a prospective patent holder. I myself know the feeling to want to beat others to patent rights, it really can make a big difference in your life. He, I feel, explores intellectualized areas, not well studied, and applies common sense ideas. He anticipates perhaps, more than verifies through research. If you have not read his patents, you should, they make fascinating reading. I browsed them this morning and it was very enlightening.
I learned very quickly here that some detest the mere mention of the man’s name. Actually though I find it all so fascinating! I have to wonder how long the arguments over this have been going on? I wish I could read some of the knock down drag out posts (smile). Are they still posted here somewhere?
Anyway, what I would enjoy discussing is the role of complement.
Oh and Sarge, isn’t it up to you to keep the peace here? Who is the topkick in this outfit anyway?
BUMP
I wish I could read some of the knock down drag out posts
This isn’t the site to find that sort of thing, as the discourse doesn’t lend itself to that, here. Sorry to disappoint you. This thread is more typical. ;-)
Who is the topkick in this outfit anyway?
The mods are Melanie, anon_22, pogge and myself. See About in the L sidebar. But keeping the peace is everyone’s business.
Thank you DemFromCt,
Are you owners also? Biologists perhpas? Are there bio’s or CV’s for the mods? Oh, and I am not dissapointed at all, I love spirited debate but only among friends. I have been at it too long to think I am always right, or even partly right, usually I feel I never know quite enough (smile). I do agree with you that civil, is the only discourse tone allowed in science. There is no place for anything but.
When I first found you here, there did seem to be a real argument which I found, quite out of context. I inadvertently got caught up and learned not to field a recombinant view (that is not my first choice ot topics). The dust of that has seemed to have settled now and it has seemed to become a genuine issue, fun.
Naplespark – at 20:35
Timing is everything, except when it’s location. ;-)
The ‘owners’ and founders are myself, Melanie and the reveres. We all share an interest; the titles mean less than the quality of the contributions. We all do political blogging, too, so we tend to be discreet about our backgrounds. The Reveres (named after paul revere, the first US public health offical) blog at effect measure.
Here’s more about Flu Wiki.
Theresa42 – at 13:23
Ok. How do they know this? I’m looking for any and all direct, experimental evidence to show that what we’re seeing are mutations and that, therefore, one can calculate mutation rates, etc., etc. As we all know, reconstructions based on phylogenetic analysis are fraught with all sorts of problems — basically, your phylogenetic tree is just as good as the data you base it on — so I’m not (so) interested in such indirect evidence.
The references you and I have found describe exactly how mutation rates are measured. The in vitro experiments are as direct as is physically possible right now and do not depend on phylogenetic reconstructions. If there were a machine that allowed us to watch random mutations in real-time, wouldn’t that be cool? It would have to be able to see through cells and somehow visualize nucleotides complexing with proteins without disrupting them. Who knows, maybe someday… In the meantime, flu scientists have the same tools available to other virologists. And they are good enought for me.
When I started to review the papers on mutation rates, I was an agnostic on the Theory of Recombinomics. But after reading the literature, I have come to the conclusion that the evidence supporting a high rate of mutation and a low rate of recombination is overwhelming. One can always wish for more information and no-one would say we have a complete information about flu viruses, but when two Theories conflict, we must decide which one is better at explaining the available data. No contest, IMO.
LMWatBullRun – at 14:19
If the error rate/mutation rate in a virus is high, and if the numbers of identical sequence data across a span of years is low, how possible is it that this is simply a stochastic event? That is, apart from the “lab escape” hypothesis, is it possible that some of these sequences are simply statistical outliers? Has statistical analysis been done on these occurances? Do we have enough data for a meaningful attempt at such? Is the granularity of the sequencing data sufficiently fine to permit detailed examination of the exact ATCG combinations? I would be very interested to see whether or not the sequences showed any evidence of minor alterations that might suggest long term residence outside a “frozen environment”
Good questions. I have wondered myself whether given the large number of sequences available, some identities are simply chance. I think it depends on how long the identities are. My guess is that Dr. Niman has provided a few examples where the length of the identities are too long to be chance, but I am not a statistician. There should be enough sequence data to address this question.
I too am looking for another explanation of the identical sequences beyond the “frozen” hypothesis. Thus far, no-one other than Dr. Niman has proposed one. So right now we are left with:
I do wish someone would come up with a third possibility.
Hi Dem,
Ahhh those pesky Bostonians, they turn up everywhere (smile).
I do not usually find the time to exchange ideas here on the net, usually my day is mired in problem solving. So this is a fun excursion. I suspect I will eventually come once in the morning in preference to reading the news, which as of late seems all bad, jeeeesh!
Anyway, I am finding my way around, thanks for the tips, and good efforts, your site is fun.
Monotreme -
The frozen virus theory may sound like conspiracy theory to some, but not this cold warrior. The Aralsk smallpox outbreak of 1971 should give one pause to consider. However, Alibek, nor any of the other former Soviet bioweaponeers has mentioned research into influenza as a weapon (at least that I can recall). Of course, the Soviets weren’t the only ones playing with bugs.
For the record, I do hope that Niman is on some level correct. It would be nice to have at least a glimpse of predictability in the future course of evolution of The Bug. I suspect that this fervent hope is the source of some of the vehemence of his acolytes. Alas, your studies don’t seem to support this proposition at the present.
The Sarge
Production of a bioweapon is not the only possible source for an escapee. I’m also considering the possibility of a poorly thought out vaccine. China, and others, have created recombinant (in the artificial sense, not the Niman sense) live vaccines that are part flu virus, part fowlpox virus. I don’t know if it’s possible for the flu genes in this recombinate virus to reassort with naturally occuring flu virus. I need to do more research.
Some examples:
Trovac Aih5, An Avian Influenza Fowlpox Vector Vaccine, As An Alternative Vaccine for Hatcheries
Immunogenicity of Fowlpox Virus Expressing the Avian Influenza Virus H5 Gene (TROVAC AIV-H5) in Cats
and this one is especially interesting:
China: New bird flu vaccine capable of prevention
Chinese scientists claim to have developed a vaccine to prevent the spread of the killer bird flu. The Ministry of Agriculture (MOA) says its new vaccine can effectively “cut a key link in the transmission chain of highly pathogenic avian influenza among water fowl.” Using reverse genetics, scientists at the Key Laboratory of Animal Influenza, affiliated with Harbin Veterinary Research Institute, altered the genome sequence of the virus to construct a vaccine that is believed to be safe for both poultry and mammals. The MOA suggests that if necessary, the new vaccine could be used on waterfowl in the high-risk area, which contains many water bodies, namely lakes and rivers.
I plan to start a thread on this when I have more info.
Tom DVM, do you know anything about this vaccine?
Great converstaion but needs to be closed due to length. Please continue here
Sarge at 22:41
The frozen virus theory may sound like conspiracy theory to some, but not this cold warrior.
Wouldn’t it be possible for all sorts of unintentional mistakes to happen in labs - pulling the wrong sample, mislabeling, or accidentally using wrong technique for the pathogenicity level of the virus being handled…lots of scenarios come to mind. Even human scientists are - well, human.
