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New WPI and CDC XMRV sequences in genbank

Discussion in 'XMRV Research and Replication Studies' started by Bob, May 17, 2011.

  1. ukxmrv

    ukxmrv Senior Member

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    Todd,

    Dr Mikovits had a look of respect on her face for Dr Coffin and whatever this paper is. She didn't have a look of being threatened by or scathing of whatever it is Dr Coffin is due to publish.

    I've seen her now at two IiME conferences and Dr Mikovits has always spoken in supportive/glowing/respectful tones of other researchers papers and findings (even when they do not match hers). Dr Mikovits was also talking to and mixing with Dr Peterson and others there freely and on good terms.
  2. currer

    currer Senior Member

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    UKXMRV is right. Dr Mikovits simply mentioned Dr Coffins paper in passing, quite respectfully and professionally. She did not give the impression that there were any issues there.
  3. toddm1960

    toddm1960 Senior Member

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    That's good to hear, I was afraid she knew of more piling on of contamination studies. As long as it's nothing more than he's said to this point.......it's good news.
  4. insearchof

    insearchof Senior Member

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    Hi Alex

    Thank you so much for this information. It just goes to show how involved these scientific processes are and how easy it is for these experiments to fail.

    So, is magnesium used to amplify RNA or is it used for other purposes?
  5. Bob

    Bob

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    Hi insearchof,

    From reading that helpful wiki entry, it seems that magnesium concentrations optimise the efficiency of the enzyme (polymerase) that is used to detect DNA/RNA in the PCR test.

    The article doesn't specifically mention RNA, but I assume that it works in a similar way when searching for RNA, but there are probably other steps in the process involved.

    If too much magnesium is used, then the enzyme (polymerase) will become more sensitive but less specific, so there will be more positive readings but there might also be a higher proportion of false positive readings. (i.e. more sensitive but less accurate.)

    And if too little magnesium is used then the enzyme will be less sensitive but more specific, so it will detect less of what you are looking for, but there would be a lower proportion of false positive readings. (i.e. less sensitive but more accurate.)

    So the magnesium doesn't amplify the RNA directly, but it can have the result of increasing the detection rates of RNA sequences, due to acting on the enzyme (polymerase) that is used to detect the DNA/RNA sequences.

    That's my understanding of it anyway, but I may have misinterpreted the article.

    I might be wrong about this, but I believe that the WPI use a less specific test, which detects a wider range of DNA/RNA sequences, but then they add another step to the procedure, which analyses the sequence that they have detected in order to determine if it is an XMRV/PMRV sequence, or not. So they only confirm that it is an XMRV RNA sequence once they have sequenced it. (Again, I might be wrong about this, but this is what I understand.) But I don't know if they work like that all of the time, for all of their studies.
  6. alex3619

    alex3619 Senior Member

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    DNA polymerase is the enzyme that actually copies single DNA it binds to. It copies that strand and turns it into another strand. Primers are small segments of DNA that can bind to recognized sequences and start this copying.

    The heat of the process is what separates the strands for potential copying.

    DNA polymerase needs magnesium to work. Too much magnesium can lead to either the DNA becoming more stable and not separating (so it cannot be copied) which can cause the reaction to fail, or as Bob commented it can alter the reaction so that it becomes easy to copy the wrong thing - magnesium concentration is critical. I do not fully understand the topic of magnesium optimization. My guess is that more than a few PhDs were earned working that out alone.

    DNA polymerase does not copy RNA. To amplify an RNA virus, it first has to be changed to a DNA strand, and this requires reverse transcriptase, an enzyme found in retroviruses.

    Bye
    Alex
  7. alex3619

    alex3619 Senior Member

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    Hi currer, something I have been thinking about is this: what is a big difference between a CFS-wise asymptomatic XMRV positive prostate cancer patient and those with ME or CFS? Our immune systems are fighting like crazy! Maybe sequence diversity will always be higher in prostate cancer patients due to ME/CFS patients fighting and destroying the virus more often. It could be that we kill and deactivate more virus, and slow its replication even further. This is just speculation, but I thought it was interesting.

    Bye
    Alex
  8. currer

    currer Senior Member

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    Hi Alex,

    I'm just coming round after the IiME conference - it has taken me almost a week to get back to normal. Sorry I could not post much earlier.
    I agree with you that we are fighting the virus, and as JM speculates, this may be protective against some of the associated cancers.

    However, when JM was discussing her research, the slide she showed had the "family tree" of all the viruses they had isolated, and how they relate to each other.
    XMRV was on the bottom branch and above were many PMLV branches, showing all the PMLV variants they had isolated so far.

    So I think that JM is simply suffering from the difficulty of getting her research published. They really are finding as many variants as they would expect for this type of virus.
    She said that we should not use an HIV model when thinking about XMRV. The true parallel is HTLV1, which does not vary much. The same virus can be found in patients forty years on, HTLV1 does not mutate much. Remember HIV has a highly abnormal replication rate - it is astronomical! (Check out the Wiki page) This is why it mutates so much. XMRV is much more usual in its replication.

    She did say that they had looked at their antibody tests. Sometimes you get an antibody to a gag protein showing up clearly, but no antibody to the expected env (this is just from memory). She interpreted this to mean that the env had mutated so that the antibody test could not pick it up. They were looking at these results in more detail as this was a sign that there was a new PMLV variant in this patient. (clever, huh!)

    This is why they are behind on some research looking for straight XMRV, because they want to be sure and isolate all the PMLV examples they find in their blood samples.

    She also quite definitely stated that active infection correlates with the biomarkers and cytokines. As I remember she said that they had found a specific cytokine profile in infection which was different to the Klimas one.
  9. heapsreal

    heapsreal iherb 10% discount code OPA989,

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    I also wonder if we have an immune defiency first and second comes all the different infections. In japan cfs is known as Nk dysfunction syndrome, or something like that. Although not 100% perfect, but the only real known cure for cfs is ampligen and it works through strengthening our immune system, it doesnt fight any particular virus or infection but strengthens our immune system as a whole to fight these different infections.

    Maybe we/they should be looking at ways to strengthen our immune system and or nk function as this is the most common immune abnormality. Also instead of trying to resurrect ampligen, maybe they should be looking for an alternative, one that is easier to use and administer then twice weekly ampligen IV's. Secondly go after co-infections with antivirals, antibiotics etc but the immune system should be first. Maybe then we wouldnt being arguing about 'the' infection causing cfs.

    cheers!!!
  10. alex3619

    alex3619 Senior Member

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    Hi, I wonder if we shouldn't be more active in promoting HTLV as a model. This has been a better model for a long time, but so many have never heard of it but most know of HIV. Like HTLV, many carriers seem to be asymptomatic. Indeed, if the prevalence of XMRV carriers is around 7% and the prevalence of symptomatic carriers is like 1 or 2% of the population, this would look a lot like HTLV.

    currer, it is good to know that the range of virus being found includes full PMLVs.

    heapsreal, fegarding ampligen, I was not aware that anyone was cured. I do know that many have periods of significant remission in symptoms, but these return. If anyone knows of someone who was cured, do you a link or reference? This would be an important step. It is however a drug with a long history of successful treatment, no other highly successful drug has been studied for as long.

    Bye
    Alex
  11. heapsreal

    heapsreal iherb 10% discount code OPA989,

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    hi alex,
    probably in my dodgy wording of cure , not 100% perfect but should have said the closest we have come to a cure and i would take a significant remission of symptoms while on the drug as close enough. I would grab it with both hands. Im thinking like HIV patients being almost cured on arv's and they also go backwards without their meds. HIV people have alot better quality of life and longevity on arv's, so im refering to a cure like this.

    But what Im getting at is going after fixing the immune system first, which they have had the best success with ampligen. I just think that ampligen has been around along time, surely someone out there has made improvements on this type of treatment by now. Then maybe marrying up this type of treatment with going after the infections we know we have will get us over the line. I think this is something that should be happening now, narrowing down the exact bug maybe secondary. I get the impression this is maybe apart of what montoya and stanford are doing by studying different infections in cfs.

    cheers!!!
  12. currer

    currer Senior Member

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    Hi Alex,
    Yes Ive added this to my last post #68 but JM is spending a lot of her time now looking for the PMLV variants in blood. This has made her behind on some of the work just to do with the XMRV virus.
  13. Bob

    Bob

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    There are further XMRV sequences submitted to genbank by the WPI.
    I've added them to the opening post:

    Whittemore Peterson Institute
    Lombardi,V.C. and Khaiboullina,S.F.
    21st May 2011


    Xenotropic MuLV-related virus isolate WPI-CI-1303 gag protein (gag) gene, partial cds
    http://www.ncbi.nlm.nih.gov/nuccore/JF907633.1

    Xenotropic MuLV-related virus isolate WPI-CI-1304 gag protein (gag) gene, partial cds
    http://www.ncbi.nlm.nih.gov/nuccore/JF907634.1

    Xenotropic MuLV-related virus isolate WPI-CI-1310 gag protein (gag) gene, partial cds
    http://www.ncbi.nlm.nih.gov/nuccore/JF907641.1

    Xenotropic MuLV-related virus isolate WPI-CI-1313 gag protein (gag) gene, partial cds
    http://www.ncbi.nlm.nih.gov/nuccore/JF907643.1

    Xenotropic MuLV-related virus isolate WPI-CI-1314T gag protein (gag) gene, partial cds
    http://www.ncbi.nlm.nih.gov/nuccore/JF907644.1

    Xenotropic MuLV-related virus isolate WPI-CI-1315 gag protein (gag) gene, partial cds
    http://www.ncbi.nlm.nih.gov/nuccore/JF907645.1

    Xenotropic MuLV-related virus isolate WPI-CI-1316 gag protein (gag) gene, partial cds
    http://www.ncbi.nlm.nih.gov/nuccore/JF907646.1
  14. eric_s

    eric_s Senior Member

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    Great, i hope some of them will sink Coffin's hypothesis (in case the ones already uploaded were not enough to prove this hypothesis wrong).

    I'm really looking forward to the day when this crazy controversy is over and we can just focus on getting help for the people affected with ME/CFS.
  15. Jemal

    Jemal Senior Member

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    Same here. I have the feeling though the controversy will probably last for some time yet... I am not a generally positive person though, so I hope I am wrong.
  16. eric_s

    eric_s Senior Member

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    I also think we might have to wait a bit more, but nevertheless there are interesting new results coming out every now and then.

    I think we can use that time to strengthen our community, make new connections, try to improve the strucutes etc. so that we will be ready to shower the good reserach with more money, gain more influence with politicians/officials, get more awareness in the public and so on.
  17. joshualevy

    joshualevy

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    TWIV #136 has a discussion of the sequences in genbank. (I'm not sure about all of them, but certainly the first batch.) The summary is simple:
    • The tiny differences between the different sequences provide strong evidence that what was found was contaminant.
    • Far from helping the WPI cause, they provide direct evidence that the XMRV found was a contaminant.

    I must say I have never heard any Virologist who did not work for WPI, say anything positive about those gene sequences. Everyone who is knowledgeable in the field says the same thing: they are evidence of contamination.

    BTW, This Week in Virology #136 is well worth listening too.

    Joshua (not Jay!) Levy
  18. Bob

    Bob

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    The Coffin paper has been challenged by the new sequences in genbank - Both Switzer's and the WPI's newly published sequences.
    Many people probably are not aware of the new sequences, because a high profile paper hasn't been published about them.
  19. joshualevy

    joshualevy

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    The TWIV #136 comments were in June, and the new sequences were in May, so I think their comments covered those sequences. Are you saying there were sequences added in June?

    Joshua (not Jay!) Levy
  20. Bob

    Bob

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    I'm saying that most people are only aware of the 3 sequences that the WPI originally published.
    The latest sequences were published on 21st May.
    A professional analysis has only been carried out, and peer reviewed, in relation to the original WPI sequences, and not the recent sequences, as far as I am aware.
    The information and analysis, that I have seen, suggests that the new sequences do have a greater variety than the original sequences.

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