Tuesday, June 14, 2011

DUSTY MILLER'S
XMRV STUDY

Dr. Dusty Miller gave this statement to CFS Central about his XMRV study:

"Our paper is in press in the Journal of Virology, and should be available online through the Journal website on Wednesday this week.  We performed our study independently of the Paprotka et al. (including Coffin) study recently published in Science, but the results do overlap.  Essentially, we found an endogenous retrovirus (mERV-XL) in NIH/3T3 cells, a commonly used mouse cell line, that is virtually identical to what Paprotka et al. are calling PreXMRV-2.  You can find both sequences on the NCBI website.  One of the points we make is that all of the PCR primers used to detect the XMRV gag region can amplify a sequence identical to XMRV from NIH/3T3 cells, which are present in many labs.

"Unfortunately, we did not find an intact copy or the right half of XMRV in any of the mouse cell lines or tissue that we analyzed, but clearly we did not look hard enough.  Paprotka et al. firmly established the origin of XMRV from nude mice.  We were pursuing the same hypothesis, but could not get early samples of the cell lines and tissues from which the XMRV-carrying 22Rv1 cells were derived."

69 comments:

  1. Could somebody please explain what this means? Are they saying they discovered another lab contaminant in a different cell line?

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  2. "Clearly we did not look hard enough". What were they looking for ? A human gammaretrovirus or contamination ? And why the continued, myopic obsession with PCR ?

    This is like letting people burn in a house fire whilst trying to work out who started it.

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  3. Thank you for continuing to cover the XMRV story, Mindy. We are very lucky to have you.

    At this point it's clear that more research is needed and the WPI are well placed to win over $100,000 in the Vivint contest, which involves voting for WPI via Facebook.

    Please, everyone, support WPI in this way so we can get some answers. Details are on WPI's homepage or here:
    http://forums.phoenixrising.me/showthread.php?12310-for-WPI-%E2%80%93-vote-daily-now-in-Vivint-Round-2!

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  4. Dusty Miller to Mindy Kitei:

    "Unfortunately, we did not find an intact copy or the right half of XMRV in any of the mouse cell lines or tissue that we analyzed, but clearly we did not look hard enough."


    I find it preposterous that Dusty Miller would say this--to a journalist! It is like saying--"I only found half the proof for my hypothesis, but I still think I'm right. My work is garbage, but so what? It's just because I didn't look hard enough. I'm sure it's really there, so I'm publishing my hypothesis as if it were proven anyway."

    Patricia Carter

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  5. The same question keeps coming to my mind. When trying to explain away XMRV as PCR testing contamination how is all the other evidence ignored. The Culturing, the antibodies and the fact that when patient samples and controls are tested and handled in the same manner the patient samples consistently test much much higher positive than controls.
    Possible PCR contamination just does not explain away these facts.

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  6. If this wasn't OUR Living Nightmare this would make a funny stupid scientists movie or documentary with all of these half-a*^ done tests.. I think they have just about ALL Proven that their PCR's are NOT adequate and many are contminated..
    Please for God Sake do a Fricken REAL Replication
    Study ~ That means also culture and Serology using 3 labs and it takes about 1.5yrs with a good sized group.
    Either do it RIGHT or Shut the F - UP..
    They just must like to see their names in print even if it makes then look like bloody fools..
    *Sigh*
    Thanks Mindy.. Keep documenting....
    *Hugs* from SeroPositive antibodies here..

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  7. It looks like the publication of this study has only been made possible because of yet another monumental failure of the peer review process.

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  8. Meanwhile, MIkovits (which she mentioned in a talk) and Ruscetti (mentioned during an FDA BPAC meeting) are locked out of publishing their findings. And they question OUR ability to be balanced?

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  9. I just checked and there is nothing being published in Journal of Retrovirology today for Miller.

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  10. Anonymous: it's the Journal of Virology, not Retrovirology.

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  11. Yes that's what I meant. I realized what I said and was going to correct it. You beat me to it.

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  12. It's after 5pm Wednesday June 15, and the Journal of Virology is published online, and it contains nothing from A. Dusty Miller.

    Is this another of Dusty's famous hoaxes? Like the study he was doing to do with Cort Johnson and Ecoclimber?

    Patricia Carter

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  13. The paper describing our XMRV study was not posted on the Journal of Virology website today, but will likely be posted on Wednesday next week. Accepted papers are only posted once per week, and the timing of posting is somewhat variable.

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  14. I think that even if the XMRV occurred in a lab setting it is still a retrovirus and quite possibly got into the vaccine supply. I am 99.9% certain that is the source of my disease. 20 coworkers were given Heb B vaccine. All of us became ill and no one has recovered. M Bloomer

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  15. Seven of my co-workers have come down with this illness. They donate blood on a regular basis as they are unaware what XMRV or HGRV's are.

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  16. M. Bloomer ~~ Have you spoken to anyone about you and your coworkers? If so, was there any follow up on your theory?
    A concerned Fibromite

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  17. this is just amazing !!!!

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  18. Dr. Miller,

    I am a layperson. Why do you say 'obviously you didn't look hard enough', that the Coffin paper 'proved' preX1 and 2 recombined to make XMRV? He has not been confirmed yet by other labs. Would you say that all those who didn't find XMRV, unlike Lombardi et al., obviously didn't look hard enough because Lombardi proved HGRVs are associated with ME?

    If XMRV is everywhere "contaminating"/infecting everything, why can't you and these other labs find it? You can find mERV-XL, but not XMRV or the other 'parent virus'? It seems to this layperson that either your lab is incompetent and shouldn't be publishing on this data or Coffin is wrong. Please disillusion me.

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  19. Dusty are you advertising your paper?

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  20. First, a response to anonymous posted 6/16 1:31 AM. No, I am not 'advertising' my paper. Like other scientists, I and my coworkers are trying to understand the potential role of XMRV in human disease, if any. We publish our findings to document our progress. Mindy emailed me asking about our study, and I provided some details.

    Now to answer Justin's questions posted 6/16 1:18 AM. When I said that I and my coworkers didn't look hard enough, I meant that we didn't look hard enough to find the viruses that Paprotka et al. have demonstrated are present in nude mice. Our failure could be because we studied only one strain of nude mice, and as Paprotka et al. have shown, not all strains of nude mice carry the PreXMRV-1 and PreXMRV-2 viruses that they found. If we had looked harder, that is, studied more strains of nude mice, we might have been successful. They were the first to solve the puzzle we were both working on. However, we found something new - the presence of a virus like their PreXMRV-2 in another mouse strain. Our results help to confirm and extend their results; therefore we are publishing these findings. We are not trying to confirm all of the results of Paprotka et al. because we believe their methods were reliable and find no reason to doubt their results.

    In the case of Lombardi et al., many others HAVE tried hard to reproduce the findings that XMRV is present in humans, especially those with CFS, and have been unable to confirm these results. In this case, the other researchers are using techniques that are proven to be able to find XMRV if present. Lombardi et al. didn't "prove" that XMRV was present in humans, they found evidence that it was present. All scientific studies are subject to testing by other scientists, and even if many scientists all agree, we don't consider this proof that something is correct.

    Regarding Justin's question that if XMRV is everywhere, why can't we find it? - we do find it, just not in mice or humans. Indeed, I and my collaborators were the first to show that XMRV is present in the 22Rv1 prostate cancer cell line (Knouf et al., J Virol 83:7353, 2009). This cell line produces lots of infectious XMRV virus (more than 10 million infectious particles of XMRV in a teaspoon of culture medium exposed to the cells) that can readily spread to other cells, including human cells, if given a chance. This finding has been confirmed by all other labs that have looked. But, while we routinely can detect XMRV in 22Rv1 cells, we don't find an intact copy of XMRV in NIH/3T3 cells, for example, only an endogenous retrovirus that contains the left half of XMRV, that we call mERV-XL.

    Hope this helps.

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  21. >I think that even if the XMRV occurred in a lab setting it is still a retrovirus and quite possibly got into the vaccine supply. I am 99.9% certain that is the source of my disease. 20 coworkers were given Heb B vaccine. All of us became ill and no one has recovered. M Bloomer


    I got ME/CFS after a Hepatitis B vaccine also, so am very interested in this topic.

    However, this vaccine does not make everyone who gets it sick. It thus seems to me there must have been something additional going on in your cohort, that everyone would have been hit so hard.

    Where were you working at the time?

    If a hospital, what kind of patients were you treating?

    Was there any reason to believe that you might have been getting a toxic exposure from the building (e.g. Sick Building Syndrome, toxic mold)?

    Did people in the building display any symptoms at all (even mild ones like depression or headache) before they got the vaccine?

    What city was this? What year?

    Thanks very much for any information you might be able to provide.

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  22. @M. Bloomer

    Whoa! That's a very significant epidemiological event. I hope you and your co-workers are in touch with some MDs and researchers who are following this up. That could provide a lot of very valuable data for CFS research.

    - Andy Vaughan

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  23. Dusty. You haven't published a paper to talk about! There are no findings at this time!

    How very scientific of you to not test the hypothesis in Paprotka et al. They too didn't test their hypothesis. PreXMRV-1 has not be shown to have even been found. Have you considered that all the negative papers also didn't try hard enough, what ever that means. Please speak in scientific terms. The others have not clinically validated their assays and have not replicated, so they cannot claim and neither can you Dusty, that they have proven their assays can detect a known positive. Lombardi et al. evidence strongly showed that HGRVs were present in ME/CFS patients. They used 4 methods and 3 labs to do this. Are you all scared to test the hypothesis that this virus is present?

    22Rv1 has been treated with testosterone. We all know the virus will reactive with this. So it's logical to assume unsensitive assay will be able to detect virus there, but not in blood, as the titre level will have been increased enormously.

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  24. Dear Anonymous (your post of 6/16 1:46 PM),

    Although our paper is not published yet, I can still talk about it. You might not believe what I say until you see the methods and data yourself, which is fair. However, the sequence of the mERV-XL provirus we found in NIH/3T3 cells is available (GenBank JF714652.1) for your expert analysis.

    The question that Paprotka et al. and my lab group were investigating was whether XMRV was present in the patient from which the 22Rv1 cells were derived, or whether it was acquired during passage of the patient-derived cells in nude mice or during culture of the cells in the lab. Paprotka et al. have clearly shown that XMRV was not present in early passages of the patient cells, and also convincingly showed that they could detect XMRV if it was there in these early passage cells. They also identified and sequenced two proviruses (retroviruses integrated into the nude mouse genomic DNA) that are highly likely to have been the parents of XMRV. In conclusion, XMRV was not present in the original prostate cancer patient from which the 22Rv1 cells were derived, but was picked up during passage of the cells in nude mice.

    One could argue that perhaps the virus was transferred from a human infected with XMRV to the cells. To rule out this possibility, one needs only to look at the sequences of the nude mouse viruses and XMRV present in the 22Rv1 cells. I know you won't believe my conclusion here, but these viruses are simply too similar in sequence to have arisen from different sources. With your obvious scientific bent, you should be able to calculate the probability to convince yourself.

    Regarding the data in Lombardi et al., I was initially convinced by their extensive analysis, in particular, their ability to grow virus from patient materials. Indeed, we entered the CFS/XMRV field largely on the basis of these data. However, published and unpublished data now indicate that all of these methods were flawed.

    Regarding the constant accusation that no one has carefully replicated Lombardi's methods, this is not true as far as most scientists are concerned. Initial reports attempting to replicate the study did have flaws, but many later studies are convincing.

    Regarding the testosterone comment, steroids such as testosterone will slightly increase production for many murine leukemia viruses, including XMRV, but none are required for high level XMRV production. We never treated our 22Rv1 cells with testosterone or other steroids, and still found production of high levels of infectious virus from the cells.

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  25. Dusty,

    Why the narrow focus on XMRV? Do you plan to investigate the findings of Lo, et al. that persons with ME are infected with proper MuLVs?

    Stated differently, are you for some reason focused strictly on tearing down XMRV, or are you interested in helping desperately ill people? If it's the latter, could you please tell us what you are doing in this regard?

    subtr4ct

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  26. As the patient that 22Rv1 was created from is now dead, it is difficult to know if they were infected. Especially when only certain generations of the cell line are available to retest. Again, these may have become infected with XMRV through experiments with other infected cell lines, if one was to believe that the virus has polluted so many labs. Obviously this still says nothing about humans being infected.

    Paprotka et al., has NOT clearly shown that XMRV was not present in the early xenografts. They did in fact detect XMRV env sequences in the earlier xenografts. Yet, as the same primers could detect XMRV and PreXMRV-1, they arbitrarily decide it was PreXMRV-1. Hence there is no proof that the earlier xenograft do not contain XMRV. Nor is there evidence that PreXMRV-1 exists, as it was constructed from sections of virus found in NU/NU Hsd strains, and the CWR-R1 cell line. If PreXMRV-1 and 2 do exist, they still have not been shown to be ancestral to XMRV and could equally be descendants of XMRV. Hence the conclusion that the XMRV variant of HGRVs was created during xenografting is an assumption without supporting data.

    As you say, it is also entirely possible that the viruses were transfer from human to the cells. But sequence variation does not rule this out. HTLV can have little sequence variation and still be regarded as different strains. Hence this is not data with which anyone can claim supports a contamination hypothesis. The other data from the positive papers also refutes that possibility. In fact further published and unpublished data proves that those papers are not flawed.

    No one has replicated Lombardi et al. It would be fascinating to get this into a court of law, as it would be easy to prove that this has not occurred. Many scientist are appalled at these erroneous claims and also agree that they have not provided data that in any way impedes on the findings of Lombardi et al, Lo et al, or any of the positive prostate cancer papers.

    This paper proves that XMRV is androgen responsive.

    Androgen Stimulates Transcription and Replication of Xenotropic Murine Leukemia Virus-Related Virus
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812331/

    Here they demonstrate that DHT stimulated XMRV replication 3-fold. Hardly what anyone could call a slight increase. As you will know the later xenografts were treated with testosterone pellets as they were passaged through mice, thus it is irrelevant if you did anything to 22Rv1, as the viral titre in those later xenografts would already have been affected and increased considerably.

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  27. Miller stated that he had found a sequence which resembled the left hand half of xmrv which was incomplete and could not find sequences corresponding to the right half of xmrv.

    There is no way of telling whether or not that sequence was an erv. If the reagents cycling conditions and primer combinations were not exactly the same as those used in Lombardi then the study is as best useless

    If he did not use primers,cycling conditions and reagents with a provenance of success in detecting xmrv env sequences in infected people then the design is infantile.

    http://www.mecfsforums.com/index.php/topic,7889.75.html

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  28. Response from Dr. Yes to Dusty Miller's latest comment here:

    http://www.mecfsforums.com/index.php/topic,7889.60.html

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  29. Hey dr. Miller did you send an anonymous person called ecoclimber to do your bidding on patient forums without an IRB approval?

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  30. Dr Miller,

    I just want to thank you for your scientific contributions to the highly interesting XMRV "debate". In retrospect, your (and your collaborators') finding of XMRV in 22Rv1 seems to have really been a essential finding in solving (most of) the puzzle.

    Just to present you with some laughs in reward, please read the following: http://www.mecfsforums.com/index.php/topic,6506.msg75682.html#msg75682

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  31. Nothing published has indicated that any of the Lombardi methods were flawed. If Miller thinks so, he should specify which studies indicate which methods were flawed. This is a serious issue, not one about which a professional scientist should be tossing around statements without backing them up on a patient advocacy blog.

    They are not accusations. Nobody has replicated Lombardi et al's methods. Period. That is a statement of simple fact, based on the longstanding scientific definition of replication. It is not a statement that can be qualified, or subject to nuance, or opinion. If Miller or "most other scientists" (how many did he ask? ) think that changing all key variables constitutes replication one must ask why they hold the scientific method in such contempt Did he ask any biochemists or physical chemists, both of whom would be appalled by the idea of not precisely replicating a PCR assay's reaction conditions and calling it "replication" or "good enough"?) .Miller and co are certainly departing from the standards demanded in biochemistry and chemistry. I cannot imagine a chemist or a biochemist viewing a PCR assay attempting to replicate another PCR assay but using different reagents reactant concentrations and coenzyme concentrations as being good enough. Such a flagrant departure from the scientific method and the laws of chemistry would provoke scorn and outrage,

    I am not sure what the original comment was, but of course the issue with testosterone was its administration to early xenografts, which could have raised XMRV viral titer in the xenograft tissue from below the assay's limit of detection to a detectable level later on in the process, thus creating the false impression that virus was not present in the early passages and suddenly appeared later on.

    Patricia Carter

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  32. This article is worth reading. It theorizes a possible relationship between vaccinations and disease (autism, CFIDS and possibly some cancers):

    * the practice of passing live viruses through animal cells in order to create live, attenuated virus vaccines started in the 1930s

    http://www.ageofautism.com/2011/03/a-theory-of-xmrv-creation.html

    * the practice of passing live viruses through animal cells in order to create live, attenuated virus vaccines started in the 1930s
    * the first autism patient was born in 1933
    * the first outbreak of CFIDS happened in 1934

    The as yet unproven theory is:
    * XMRV is the result of bad vaccine creation practices where two common mouse viruses combined to create the XMRV retrovirus
    * XMRV has been in many vaccines since the 1930s
    * XMRV causes autism in some people
    * XMRV causes CFIDS in some people

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  33. RRM, great find. Omerbasket is entirely correct:

    "Gerwyn, please, don't jump at me - I really hope that you are on to something, but I have a question: VP62 and XMRV from 22Rv1 are 99.8% identical, at least if the BLAST that I did is good enough. Also, if I found the envelope sequences correctly, the envelope sequences of VP62 and XMRV from 22Rv1 are almost identical - they have only about 2 or 3 nucleotides that are different, out of 1937 nucleotides."

    "So how is it possible that "the 22 RV1 "xmrv" env protein is considerably different to the XMRV isolated by Silverman?""


    But Gerwhine keeps on insisting that only 30% of the 22Rv1 envelope has been sequenced:

    "if youl ook at knouff et al Omer it is clear that only 30% of the env region has been sequenced. Yes it is possible that the env sequences have terminal deletions"

    What Gerwhine doesn't realize is that, while Knouf et al. only provided part of the sequence of the XMRV virus present in 22Rv1 cells, Paprotka et al. (J Virol 84:5719, 2010) did completely sequence the XMRV virus present in 22Rv1 cells, and this is the "complete" sequence present in GenBank.

    An excusable mistake if Gerwhine didn't trash the opinions of all experienced virologists and portray himself as knowing all the answers.

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  34. I think Dusty posted another comment here, but it appears to be gone now. Any explanation? Maybe I'm just misremembering...

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  35. I must apologize for my comments about Gerwyn of 6/16 at 6:14 PM. I didn't realize that the thread I was referring to was from March of 2011, and likely Gerwyn has recognized his mistake by now.

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  36. Dusty Miller said...

    But Gerwhine keeps on insisting . . .
    -----------------------------------------------------
    You just lost any respect I might have had for you by stooping to name-calling, Dr Miller.

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  37. In response to Anonymous dated 6/16/2011 4:25 PM

    "Hey dr. Miller did you send an anonymous person called ecoclimber to do your bidding on patient forums without an IRB approval?"

    This question relates to a clinical trial I was trying to initiate to determine whether XMRV was present in human subjects with CFS. I wanted to see if I could confirm that subjects who had tested XMRV positive were indeed positive. Because I am a retrovirologist with limited knowledge of CFS, I linked up with Ecoclimber, who has CFS, to help design the criteria for patient inclusion in the trial. We thought it a good idea to determine whether subjects with CFS might be interested in enrolling in such a trial, with the understanding that we would require IRB approval before initiating a trial. It turned out that many potential trial participants only wanted to enroll in the trial if we were sure to find XMRV, an unacceptable condition.

    Regardless, I applied to my institutional IRB for permission to perform the trial, requesting that I be able to inform participants of the results. I reasoned that if I had CFS, I would want to know the results. The IRB committee felt that I should not provide results to participants, and denied my application. They felt that CFS subjects could not handle the information.

    I reapplied, agreeing to their insistence that I not provide results to patients, but they again denied my application, now insisting that I involve a population scientist in my study, and expressing concern that the risk/benefit ratio (risk of blood draw/information gained about CFS) of my study could not be determined. I gave up on the study in disgust over the IRB's conditions, and over concern that some CFS subjects only wanted to hear that they had XMRV.

    Our decision to drop the clinical trial proposal turned out to be a blessing in disguise, as we almost certainly would have been scooped by the recent paper by Knox et al. in Science Express.

    One of the members of the ME/CFS Forums did contact our institutional IRB, formally claiming that my activities on the web constituted a violation of human subject rules. After investigation, we were absolved of any violation of the human subjects rules. I have the right to discuss a proposed trial with possible participants, provided that I don't actually begin the trial before obtaining IRB approval.

    So let's move on.

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  38. Hey Bubbles, my bad, sorry about the name calling.

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  39. How can you take any researcher seriously when he engages the help of a website owner that is making money off ME/CFS patients, and a non entity forum member named Eco? Miller had these two try to recruit XMRV positives off a patient forum with no IRB? Not too professional. Here's the proof: http://www.facebook.com/notes/xmrv-global-action/response-from-dr-dusty-miller-on-pending-xmrv-study/494784121796

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  40. Dear Dr. Miller,

    I've had CFS for 28 years. Thank you so much for your interest in XMRV and your contributions. Perhaps you can comment on this:

    http://www.retrovirology.com/content/8/S1/A234

    If not now, perhaps when and if this is peer reviewed and published.

    Personally, I forgive you for slipping and resorting to name calling when it comes to the insufferable Gerwyn. He just wastes people's time. You are very gracious and patient to come here to this blog and answer all these comments. I for one appreciate it.

    Nicola

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  41. Dear Nicola,

    With regard to

    http://www.retrovirology.com/content/8/S1/A234

    I would need to see the final paper to accurately interpret the results. But, given the problems that many have documented regarding contamination of PCR assays with mouse DNA, I am suspicious of PCR results showing sequences that are close to those found in mice. But, you might ask, what if the viruses came from mice, wouldn't they be expected to match mouse virus sequences? Possibly, but many endogenous mouse viruses are unable to replicate because of mutations and deletions that accumulate over time in endogenous retroviruses, and therefore would be unable to spread to humans. In addition, humans have multiple defenses against mouse viruses that mice do not have, making it difficult for mouse viruses to gain a foothold in humans.

    The key results I would like to see are the sequences and properties of the viruses that the authors claim they can grow in the LNCaP (human prostate cancer) cells exposed to the human plasma samples. Are these viruses all the same? Do they match known mouse viruses? Do they match any viruses present in the authors' labs? If I were reviewing the paper, I'd insist on some virological characterization of the cultured viruses. So far, all of the data appear to consist of PCR analyses.

    Anyway, we need to see the final publication.

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  42. Miller this is not true. Ecoclimber is not a patient. He is someone selling what he calls a cure for CFS. Cort Johnson is a modern day snake oil salesman who would not even allow you to recruit patients off his website (thinking liablility). But Cort had no problem helping you by disguising himself on other forums to try and sucker patients in. Not a very good action for someone receiving funding as a nonprofit in the names of CFS patients. I'm sure both of them had to benefit somehow with helping you try to obtain XMRV postives when you had no IRB. What was in it for them? I call these actions very unprofessional both for a nonprofit and a researcher. And noone wanted to buy ecoclimbers supplements.

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  43. And it wasn't that patients expected to be found XMRV postive. We already know. How could you be trusted after trying to conduct a study using a non-entity forum member named Eco and Cort Johnson, a website owner. And Ecoclimber said he was providing the funding but was lying too. There are professional ways to conduct scientific studies. That was not it.

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  44. Dr. Miller, thank you very much for taking the time to answer people's questions. If possible, I would like to ask one as well-

    One aspect of the WPI's XMRV study was the use of various antibodies to detect XMRV (or at least that was what was concluded by the authors). There is a quote by Harvey Alter in which he states that most of the CFS patients in his study also had antibodies whereas most of the controls did not, although no information was given on exactly what antibodies he was referring to/using. I realize that some of this is unpublished information and therefore you cannot comment on it, but according to a recent abstract entitled 'Prevalence of XMRV in blood donors, HTLV and HIV cohorts', it states that "XMRV seroprevalence ranged from 0 - 0.6% in US blood donors, HIV-1 infected and HTLV uninfected subjects. Notably, 4.1% of Japanese HTLV-I infected individuals were p15E reactive. Inspection of sequence homology between HTLV and XMRV revealed a high level of conservation within the immunodominant region of HTLV gp21 suggesting increased seroreactivity is due to cross-reactive antibodies."
    http://www.retrovirology.com/content/8/S1/A222

    My question is would it be possible to analyze the findings of the antibody portions of the above XMRV/MLV studies and see what the antibodies were reacting to, if not XMRV/MLV's? There was a UK study by Groom et al. which showed that more controls than patients had "XMRV" neutralizing antibodies, so various antibody findings are not necessarily conclusive in and of themselves depending on the situation, but with the high probability of some known and/or unknown virus/viruses making at least subsets of CFS patients sick and with some of these antibodies cross-reacting with other retroviruses, are the antibody findings themselves not worthy of more exploration?

    Just trying to salvage whatever can be salvaged out of this whole thing, thank you very much again for your time and expertise.

    Sincerely,
    John

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  45. Dusty, how is it that you don't recognise all the assumptions in Paprotka et al., but have repeated their conclusions. Are you not an independent thinker? How can you claim they have found the origin without proving the existence of PreXMRV-1? Without knowing which mice were used in xenografting. Without addressing the points I raised earlier. Also, they did detect XMRV env sequences in those earlier xenografts, which kills their belief in the power of contamination.

    HGRVs as you know Dusty, are not mouse viruses. So perhaps its time to stop repeating that others have had mouse contamination problems, as the labs capable of detecting HGRVs have had no such issue. They also didn't only use PCR, results for which you have no explanation. What you need to do, if you decide its not too scary, is get a set of fresh patients, absolutely no need for retesting. Don't want to have only the same handful of people found positive after two years and no progress with a virus that may be infecting such a large section of the worlds population. With the WPI and NCI now finding 99 of the original cohort positive, use of correct participant selection criteria and replication of methodology will yield the correct results. You wouldn't want to go into a paper not knowing if your assays work now would you?

    XMRV has been found integrated into humans and no clone can enter a cell. The infected cell lines have never been in the WPI, as the CDCs testing also proved. The question of course is whether your independent PCR variables have the same values as their counterparts in the nested reverse transcriptase assay designed by Urisman et al. and adapted by Lombardi et al. Dusty, as you did not detect a whole provirus or even a full half, you have no idea what you amplified or whether or not it is truly an ERV. Assumptions should not be presented as fact and I hope this does not appear in your paper, like it did in Paprotka et al. It would be interesting if the cells in question are investigated using PCR assays capable of detecting XMRV env sequences and the PCR assays of Lombardi or Lo, which detect XMRV gag. This would avoid the guesswork science we are being exposed to at the moment.

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  46. To Anonymous of 6/17 12:46, 2:19, and 2:30 AM.

    What exactly is your problem? Hiding in anonymity, barfing up the same tired story. Your Facebook proof of our criminal activity clearly states that

    "The study has NOT yet been formally announced - it will be announced once IRB approval has been received".

    Ecoclimber has ME/CFS. I have met him in person, and he truly wants to find a cure for the disease, be it caused by XMRV or something else. He did offer to help fund the proposed trial. I have very little funding for my XMRV work, and I welcomed his assistance. You and others like you have hurt him deeply by your accusations.

    Cort offered to help spread the word about the study, to provide information to patients about the study design for their feedback, and so they ultimately could decide if they wanted to participate.

    Why don't you come out of the shadows and tell us all what your hidden agenda is?

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  47. @M. Bloomer

    Whoa! That's a very significant epidemiological event. I hope you and your co-workers are in touch with some MDs and researchers who are following this up. That could provide a lot of very valuable data for CFS research.

    - Andy Vaughan

    While I appreciate your noticing that this report by M. Bloomer is something that should be rigorously investigated, I have to wonder about your grasp of M.E. history. This is what we've been up against since the early 80's and arguably much earlier than that.

    We are an epidemic hiding in plain sight, with most of us receiving little or no treatment. Our history is marked by significant cluster cases that have been ignored. What epidemiology the illness has received, with the very notable exception of the estimable Lenny Jason, has hovered between the risible and the outright destructive because of self-serving disease definitions manufactured and implemented by the CDC and a small, but influential group of U.K. psychiatrists.

    Why this has happened, well, you could write a book. In fact one has been written: Oslers Web, by Hillary Johnson. And as remarkable as that book is, I'm sure there's another book to be written fleshing out even more problematic dimensions of why and how this epidemic was ignored.

    Your naivete would be touching if it wasn't reflecting an oblivion that has been responsible for enormous and unnecessary suffering.

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  48. as for the your ASSumption that all of us with cfs want to take part in only xmrv-positive studies - you are so off base it would be laughable if it didn't cost the patients so dearly. i have had this illness since 1980, and i don't care if they find it's xmrv or vaccines that cause it - i just want someone to take seriously the fact we are suffering and are untreated -all because the ins co's and ss doubt they can handle the extra claims. well we're all paying the price now - it's spread like crazy while all of the medical staff blindly follows the cdc's idiotic stereotyping. well done all of you medical "PROFESSIONALS"

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  49. i did not make clear - above comment is intended for D. Miller - sorry for the lack of clarity.

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  50. Dear John, (post from 6/17 5:46 am)

    Your idea of using antibodies present in CFS patients' blood as a method to track down viruses that might be causing the disease is a good one in principle, but perhaps difficult in practice. The problem is that people have antibodies to all sorts of viruses in their blood, making it difficult to know which are reactive with the viruses that might be causing their disease.

    A better approach might be to look directly for a virus that is uniquely present in CFS subjects. The virochip method used to first identify XMRV in prostate cancer is a good example, even if the results in this case were misleading. What would be done is to take DNA and/or RNA from patient blood samples, and hybridize this material to the virochip, which contains DNA that matches conserved sequences in many types of viruses. See which spots light up in many CFS subjects, compared to controls, and you've got some leads to explore.

    Another way to look directly for such viruses would be to perform what is called "deep sequencing" on DNA and/or RNA from patient blood samples, to try and find viral sequences that are present specifically in patient samples and not those from control subjects.

    Unfortunately, many common viruses would show up using both techniques, making the results somewhat difficult to interpret. Also, these techniques are relatively expensive. But perhaps taking patient samples from a cohort of subjects that all got disease at the same place and time would help identify a particular virus involved in the outbreak. For example, a group of people apparently getting CFS after a Hep B vaccination could be sampled, under the hypothesis that they all got a particular virus present in the vaccine, sequences from which should show up in all of the vaccinees.

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  51. Dusty, the serology assay in Lombardi et al. was unique to the SU protein of MLV viruses. It could only be identifying an immune response to an MLV virus infection, not a HERV or a mouse ERV or a virus other than an MLV. It cannot be a virus other than an MLV.

    You have no evidence that there was anything wrong with the virochip method in Urisman et al. Nothing to use to claim the results were misleading.

    As we cannot say how or when ME/CFS is caught, it is impossible to pretend that you would be strickly able to look at those who got sick and those who didn't. Especially if you insist on using sociopolitical definitions of fatigue.

    Why are you suggesting the virus is even in vaccines. The only hypothesis at the present time is that vaccine may reactivate the XMRV already present. Are you attempting to disparage the ME/CFS community?

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  52. Response to post by Anonymous on 6/17 1:21 PM

    your paragraph 1 - Try to think outside the box. The cause of CFS might be a virus other than an MLV. Humans are naturally resistant to MLVs, so this type of virus is not likely to be the cause of CFS.

    paragraph 2 - Have you not seen the papers from many groups who have been completely unable to find XMRV in prostate cancer? Aloia et al., Cancer Res 70:10028, 2010 comes to mind.

    Paragraphs 3, 4 - Haven't you read the thread above? To quote:

    ">I think that even if the XMRV occurred in a lab setting it is still a retrovirus and quite possibly got into the vaccine supply. I am 99.9% certain that is the source of my disease. 20 coworkers were given Heb B vaccine. All of us became ill and no one has recovered. M Bloomer"

    and

    "Lisa Petrison said...

    I got ME/CFS after a Hepatitis B vaccine also, so am very interested in this topic.

    However, this vaccine does not make everyone who gets it sick. It thus seems to me there must have been something additional going on in your cohort, that everyone would have been hit so hard."

    Do you think these posts are an attempt to disparage the ME/CFS community?

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  53. Dusty, try and stick to the scientific method. The serology results can only be to an MLV virus. Humans have not been shown to be resistant to MLVs and no other hypothesis for another virus as the cause of ME/CFS exists. This data from Lombardi et al. has never been challenged.

    I have seen all the data from all the published papers and none of it refutes the findings in any of the positive papers. It cannot, because the assays were not validated and no replication study has ever been undertaken. Paprotka et al. is a jolly old story, but lacks data, relying far too much on speculation.

    If you have evidence that is not anecdotal for vaccines being infected with HGRVs please present it here now. Otehrwise you are assuming those people became infected at that moment the vaccine was administered and not entertaining the alternative explanation that it reactivated a previous infection.

    Again, you pointing to these stories may be cute, but you have no other evidence.

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  54. Dusty, congratulations in becoming another of those contamination fanatics. All you can do is cling to your immaculate contamination like religious zealots, but refuse to put any data to the test. That is not science.

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  55. Hey you, Anonymous, arbiter of the Scientific Method! Come out from behind that tree so I can see your credentials. What, don't want to show yourself?

    Do you list yourself as Anonymous on your scientific papers? I'd like to look up your work.

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  56. You have nothing to say about the details do you Dusty. Can't argue the details can you.

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  57. Dusty, exactly what is PreXMRV-1? Where does it come from? How does it combine to form XMRV?

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  58. He won't answer that after avoiding the issue in his post up until now.

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  59. Dear Anonymous,

    You tiresome idiot, PreXMRV-1 is an endogenous retrovirus found in nude mice. The complete sequence of PreXMRV-1 is available from GenBank, accession number FR871849.1. Read the recent Science paper from Paprotka et al. to see how it recombines to form XMRV.

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  60. PreXMRV-1 was found using primers that are also capable of detecting XMRV. Therefore they have no reason to claim the existance of PreXMRV-1. What they threw together has only been labelled PreXMRV-1, but may infact be XMRV sequences + other seqeuences from other viruses.

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  61. Dr. Miller, what proof do you have that PreXMRV-1 exists?

    Quote: PreXMRV-1 was found using primers that are also capable of detecting XMRV. Therefore they have no reason to claim the existance of PreXMRV-1. What they threw together has only been labelled PreXMRV-1, but may infact be XMRV sequences + other seqeuences from other viruse.

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  62. No, it stinks of another shoddy, quick and dirty study meant solely to kick out more "proof" that this third retrovirus does not exist.
    Look under the rocks and bullshit_t to find the strong and controlling hands of the US and UK governments. The "whomevers" within both governments are terrified and working over time to kill this retrovirus off and all further research. But we won't let that happen this time. Will we?!

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  63. According to Miller we don't know if 22Rv1 cell line is infected with XMRV, as only 30% has been sequenced and assumed to be XMRV. Miller is therefore relying on his opinion and not evidence.

    If we only ever use our experience and stick rigidly to the same mould when presented with new data, then progress will never be made. Flat earthers' spring to mind.

    Putting these questions to the test should be the goal of any professional scientist. So where in Knoff et al. (2009) does Miller get this idea for?

    "The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, most frequently in humans with a defect in the antiviral defense protein RNase L, suggesting a role for XMRV in prostate carcinogenesis. However, XMRV has not been found in prostate carcinoma cells. Here we show that 22Rv1 prostate carcinoma cells produce high-titer virus that is nearly identical in properties and sequence to XMRV isolated by others and consist primarily of a single clone of cells with at least 10 integrated copies of XMRV, warranting further study of a possible role for XMRV integration in carcinogenesis."

    22rv1 needs to be investigated again, as VP-62 is now considered capable of contaminating samples.

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  64. Well this should take away the doubt.

    http://www.ncbi.nlm.nih.gov/nuccore/FR872816.1

    XMRV complete proviral genome, isolate S-162
    GenBank: FR872816.1

    AUTHORS - Sabaliauskaite,R., Bulavaite,A. and Lazutka,J.R.

    TITLE - Xenotropic murine leukemia virus-related viruses in Lithuanian prostate cancer patient population

    JOURNAL - Unpublished

    REFERENCE - 2 (bases 1 to 8562)

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  65. Yes, it does. If true, it definitely shows that any study which shows absolutely no significant variation in sequences collected around the globe (like Lombardi et al.) is contamination. The HTLV-1 hypothesis doesn't fly.

    Think people. Think.

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  66. Wrong try again.

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  67. The new isolate in the GenBank is 95% similar to the VP62 strain of XMRV. It meets the requirements to be called XMRV.

    You don't say why you believe that XMRV is not like HTLV, but the man who discovered HTLV, Frank Ruscetti, disagrees with you. He says it is not contamination.

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  68. It's really very simple: if true (i.e. if this new sequence is indeed a strain of XMRV) then you would have several full isolates from the WPI that are ~99% identical to a lab clone (and each other), even more partial sequences from WPI that are also very similar (98-100%) to this lab clone (and each other) and one (1) isolate from another lab that suddenly shows ~5 times more diversity than this one lab in the USA has shown (from samples around the world).

    If that one sample "is true", it strongly supports the notion that these other strains are lab artifacts. The HTLV hypothesis, that the lack of diversity can be explained by a slowly evolving virus, really does not fly then.

    Of course all of this is merely academic because the newly found strain does not branch with known strains of XMRV on a phyologenetic tree.

    Your appeal to authority is misplaced by the way. The cumulation of all authority on a subject within science is called consensus. As the consensus opinion is not on Ruscetti's side, we can safely conclude that lots of world class experts are disagreeing with you instead of with me (which doesn't prove that I am right by the way). I've explained this more extensively here by the way: http://www.virology.ws/2011/05/06/ian-lipkin-on-xmrv/#comment-200063700

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  69. The new isolate is XMRV and from Lithuania.

    It is not from the US, so why would anyone believe it should be as close to VP62 as the WPI sequences? Also, people will, like mice, have other strains and variants within them.

    The new CDC sequences also fall between this new isolate and the ones that were previously in the GenBank.

    Where do you get the idea that this supports contamination? It rules it out. None of those new sequences come from a cell line. The model of HTLV is very appropriate due to the cells XMRV prefers.

    Since when does a XMRV have to be near to one strain of XMRV? Making it up as you go along.

    There is no thing as consensus science and actually you would find there are about the same number of retrovirologists who are backing Ruscetti's position. It gets easier the more of them find the virus in people. Your link is incoherent.

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