TWiV 113: Alan Rein on XMRV

26 December 2010

Hosts: Vincent RacanielloAlan DoveRich Condit, and Alan Rein

rich conditOn episode #113 of the podcast This Week in Virology, Vincent, Alan, and Rich discuss the retrovirus XMRV with retrovirologist Alan Rein of the National Cancer Institute.

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  • RRM

    Very nice TwiV, thank you for that.

    It seemed to me that the Hue paper made a pretty convincing point however, which the participants in this TWiV didn’t fully address. Even you cannot just assume that XMRV “behaves” like HIV (or other viruses), I still wouldn’t expect more diversity in a lab than in cases around the world. As Hue et all wrote in the Retrovirology paper:

    “Even under the most conservative hypothesis that XMRV undergoes almost no evolutionary change upon transmission, we would expect sequences sampled from geographically-disparate individuals, with no known epidemiological linkage, to exhibit more diversity than sequences derived from a single infected cell line.”

    Although definite proof of contamination of those positive samples can not be provided this way, it certainly supports the argument of contamination in a pretty convincing way, in my opinion. During the TWiV, all members were hesitant to embrace this argument of Hue but didn’t really argue why.

    Can you or anyone else explain why it is not such a convincing argument after all? How can one reasonable explain that the XMRV in infected people around the world contains less diversity than the XMRV derived from a single cell line in a lab?

  • Anonymous

    12

  • Anonymous

    Hue et al. claims that there is no significant variation between the viral sequences isolated from patients. Fully sequenced clones from two patients did display a 2% sequence variation. The gag sequences isolated from other patients show considerable variation (despite the 24 bp deletion in glycogag) and polymorphism. The same is true of pol sequences (Urisman et al, 2006). The 22RV1 cell line does contain XMRV. It is the standard positive control. The sequences of XMRV expressed in this cell line are ancestral to the sequences found in XMRV sequences recently found in prostate cancer patients. This is actually clear evidence of XMRV replicating and naturally changing over time and provides direct evidence contradicting the hypothesis that it is a laboratory contaminant.

  • RRM

    You are actually copying and pasting some standard critique that is was posted on some pro-XMRV->CFS sites (e.g. XMRV Global action and Niceguidelines blog). The critique that is posted on those sites does not seem very solid, however.

    – Hue et al observe that the sequence variation among the “wild” publicized samples is less than the variation observed in the lab. That in itself is a solid argument, I would say. Which two fully sequenced strains do contradict these findings if I might ask?

    – The fact that XMRV derived from the 22RV1 cell line is ancestral to publicized “wild” XMRV, is exactly what you’d expect from a laboratory contaminant (without actually proving that is is, naturally). It would only be evidence of XMRV replicating in the human population if the prostate cancer patient from which the 22RV1 cell line originated, is coincidentally also the source of the (supposed) worldwide XMRV epidemic, which is highly, highly unlikely.

  • Anonymous

    ‘Does not seem solid’ is no argument to suggest that the points raised are not solid. The argument proposed is excellent.

  • RRM

    You might wanna look at the bullet points, where I explain why these arguments do not seem very solid, despite being repeated on several websites which, ironically, is no argument to suggest that the points rased are valid. : )

    I’ll reprase:

    – It is merely asserted that two published seuences contradict the phylogenetic findings of Hue et al. Without naming the sequences in question, it is no solid argument to conclude that the phylogenetic observations of Hue et al are incorrect.

    – The fact that a known sequence from a commonly used cell line is the (probable) ancestor of all known “wild” XMRV, is exactly what one would expect with laboratory contamination. Without contamination, you would expect at least some “wild” XMRV to lead to a different ancestor than the sequence you happen to have in your lab. After all, what are the odds of accidentally stumbling upon the “father” of all known XMRV in the world, in some random prostate cancer tissue that you’ve checked?

  • Anonymous

    The sequenced clones from two patients that displayed a 2% sequence variation, showed little sequence variation. Other patient have shown gag and pol to be different, with much greater variation.

    The 22RV1 cell line is XMRV positive, and is the standard control. That is not evidence of contamination. Why would you expect more recent samples to predate that from a prostate cancer patient from 1993?

  • Robw
  • Lauren

    A very good podcast.

    Alan says, XMRV could be a mistake and not in humans at all.

    2011 is going to be an interesting year.

    I feel a lot of sympathy for all XMRV+ if in fact they do not have a retrovirus after all.

  • Sarahphilips231

    I do not believe that XMRV is a ‘passenger virus’ as suggested.
    We know from Bob Silverman / Steven Goff that the retrovirus XMRV has an on and off switch. (Androgen and hormone responsive elements)
    Please see the attached charts.

    http://i1188.photobucket.com/albums/z403/xmrvposter/Untitled-2.jpg

  • http://pulse.yahoo.com/_N3WMFLBVFXKFYSAIY7RFD77ZZU Kermit t

    Heparin tubes, water, phosphate buffers, anything could theoretically be contaminated. But in the real world all these things would affect controls and patients equally. And in properly blinded experiments you cannot have “more handling” of patient samples.

    John Coffin’s theoretical experiment regarding a swimming pool and mouse DNA also would not be true in reality. It is about time that idea was kicked into touch.

    He should actually try doing it before he makes such comments from ignorance. In reality molecular distribution and chaotic behaviour prove that the actual distribution of DNA within a swimming pool at the limit of detection will actually result in a failure to detect any DNA. The point is it cannot mix uniformly! Now should John Coffin do the experiment and find that 10^3 or 10^4 more contaminant would be needed, that would be a mighty blow to the contaminant theorists. What we are dealing with is the real likelihood of contamination in a real lab – not thought experiments.

  • renethorpe

    blood from people with CFS was used to infect the monkeys. would a contamination have produced the results of this study

  • Anonymous

    I too would like to see those who are getting negative results actually attempt to do a replication study of Lombardi et al.

  • Anonymous

    Could be a mistake, could be a contaminant. Yes, that is a possibility, but the evidence strongly suggests that is not the case. Unfortunately if MLV-related retroviruses are not associated with ME, which again the evidence doesn’t really support, what will happen to research into the disease? My guess is more funding for psychosocial studies, nothing for biomedical research, and perhaps even an attempt to reclassify this neurological disease. It’s a terrible situation, and no one in authority has been monitoring and checking that prejudices are not hindering real progress towards a cure. I’m not for one moment suggesting that anyone should make a false association, but that this is the reality. Therefore this MLV-related retrovirus research should be given a chance. No one should be able to claim it’s over before a through investigation has been conducted, and we are not really much further along than we were in 2009.

  • http://pulse.yahoo.com/_BYNFF3RVT3YYKQOFVSK3WCHXTI Mat

    Alan Rein’s calls for less funding make no sense. He claims he wants to prevent money being wasted as he believes that XMRV is not real. Yet Alan Rein repeats several times he has no explanation for how labs can find the virus repeatably in patients but not controls. Indeed he hasn’t even read the positive paper on prostate cancer discussed.

    It seems that those that have attained positions of authority such as Alan Rein are keen to deny scientific discovery, with little more backing than dogma or thought experiments. When once dogma and pride had Galileo hanged, in todays world funding has the stranglehold on scientists.

    Money cannot be wasted in reaching the correct answer – and it must be reached beyond all doubt for the millions that are suffering from this disease. Money is only wasted on CBT, GET. There is enough prima facia proof of the virus, that those capable of finding should be granted substantial funding to establish whether or not this is the cause of ME/CFS.

  • Nicola

    I think the new control assay for mouse DNA mentioned in this TWiV will detect contamination. I can’t remember the awkward name but it’s for a retrotransposon in mouse nuclear DNA. If patient samples are all negative for that I don’t think the contamination theory will hold. I never thought the mouse mitochondrial DNA assay was good enough, and Alan Rein discussed in this TWiV why it’s not.

    Patient samples and control samples for the science study were collected at different times and places and by different people. The patients came from selected practices and controls were from a blood bank I think. I am not saying this makes it likely at all that patient samples were contaminated by mouse DNA at collection. I’m just saying that we need a proper control, and it appears that we have that now.

  • Anonymous

    The unpublished UK study used the same collection methods.

  • http://pulse.yahoo.com/_BYNFF3RVT3YYKQOFVSK3WCHXTI Mat

    The 8 samples that were repeated in the Lo/Alter paper were also collected in the same way as controls. Furthermore the Singh study will use ~100 patients and ~200 controls all collected identically, age, sex and geographically matched. That is proof.

    Using the mouse DNA assay is not proof of anything and is certainly not suitable as a control because for all that is know it will detect some hitherto unknown molecule completely unrelated to XMRV in every sample, control and patients. Alan Rein’s comment on the mouse DNA assay is an example of an artificial obstacle.

    Proof is simply healthy control Vs patient, collected identically, fully blinded, and repeated.

  • Nicola

    Mat, I was thinking that if the patients in the Science study were negative on the new mouse DNA assay, that would mean something. One of the most important patients in that study is deceased, and if there is any sample left, I would like to see the mouse DNA assay done on that.

    Proof of what? That XMRV causes post-exertional fatigue of CFS? I haven’t heard anybody address any possible mechanism for that. Even if what you say is true, that we have 5% of controls positive, we still have no idea how this virus causes this illness.

  • Anonymous

    The WPI and Ruscetti at the NCI have talked about how this could be causing ME.

  • http://pulse.yahoo.com/_BYNFF3RVT3YYKQOFVSK3WCHXTI Mat

    Nicola, I would agree with you but for the problem that you are making certain assumptions about the Mouse DNA assay that are not true.

    – First you are assuming that the mouse DNA assay can accurately determine contaminant from XMRV in a real clinical sample – that is not proven.
    – Second you are assuming that contaminant should not be present in any level in patient or control – again not true if mouse DNA is everywhere.
    -Third you are assuming that a negative test would prove something and quell the contaminant theorists, again given that there have been multiple methods including isolation of the virus from patients and antibody responses, that would seem highly unlikely.

    All that is known is that the mouse DNA assay is extremely sensitive for mouse contaminant in the test tube. As stated above, this may indeed be the cause of more problems. It is certainly a distraction and is a significant waste of money as it proves nothing in the real world. The investigation must continue as it has – controls Vs patients studies in labs that can find the virus – viral isolation – antibody detection and then treatment trials.

  • Anonymous

    Is there a short character limit or something? I tried posting a comment a few times with nothing showing up.

  • Anonymous

    (that is odd; now that my comment went through I will try posting the original comment that never went through)

    I am a CFS patient, not a virologist, so I do not understand all of the technical aspects but always enjoy your discussion on the topic and the coverage of both sides of the story. I wanted to thank you for your balanced coverage of XMRV.

    As I am not a virologist some of these questions may be stupid or have obvious questions so bare with me. What struck me from this podcast was the prevalence of mouse DNA in water. Water is used in the manufacturing of almost everything including medical testing equipment and chemicals. Heparin contains a large amount of saline and saline is mostly water. You mentioned that the heparin in some blood vials was testing positive for XMRV, could this be from the water used in the manufacturing? What is needed to remove mouse DNA from water since you said reverse osmosis is not enough?

  • Andrew Kewley

    Of course there is another possible solution to this contamination issue:
    http://www.frontiersin.org/virology/10.3389/fmicb.2010.00147/abstract

  • Anonymous

    It could be, but I don’t think those studies are coming up negative because there is no retrovirus there. This is not to say it is there, but that there are other consideration, such as methodology, that are different.

  • lancelot

    there is definitely something wrong with trying to post here. sometimes it post and sometimes it just doesn’t show up. i had lost a couple long posts at different threads.

  • lancelot

    (this is another repost so i may of lost some thoughts and words)

    Alan Rein Summary:

    1) Alan says that Lo/Alter did not find exogenous MLV’s but found mouse contamination due to using faulty(contaminated) assays. Recommends Harvey to go back and use the IAP probe. He says the MLV sequences alter found is exactly the same as the sequences in mouse(contamination).

    2) Alan did not find XMRV in his prostate tumor study.

    3) When Ila Singh gave some of her XMRV+ samples to Alan, he found those same samples to be negative for XMRV when tested with his antiserum.

    4) Alan says mouse contamination is so abundant that another researcher found XMRV mouse contamination in different cereal boxes.

    5) Alan says it is not likely that geographical differences account for producing both positive and negative XMRV prostate studies.

    6) When asked about the XMRV antibodies found by WPI, he had no comment except to say that it must be replicated by other labs.

    7) Alan says there is mouse contamination(nucleic acid) in water that even a .2micron reverse osmosis filter cannot filter out mouse nucleic acid (contamination) from water. Thus, all the water used in the labs have mouse contamination.

    8) Alan says another researcher found XMRV mouse contamination in cereals.

    9) When asked about the antibodies to XMRV found by WPI proving infection, Alan didn’t have any comment but that it has to be reproduced by other labs.

    10) Alan says he does not know nor can he explain why patient samples have more contamination then control samples.

    11) Alan strongly says that it is a big possibility that XMRV is just a contaminant and that it doesn’t exist.

    12) Alan says if there is viruses(XMRV) in prostate tumor tissues, it is in so low a number that it doesn’t have anything to do with disease and it is just a passenger.

    Alien Rein is either 100% right or 100% wrong.

  • lancelot

    (this is another repost so i may of lost some thoughts and words)

    Alan Rein Summary:

    1) Alan says that Lo/Alter did not find exogenous MLV’s but found mouse contamination due to using faulty(contaminated) assays. Recommends Harvey to go back and use the IAP probe. He says the MLV sequences alter found is exactly the same as the sequences in mouse(contamination).

    2) Alan did not find XMRV in his prostate tumor study.

    3) When Ila Singh gave some of her XMRV+ samples to Alan, he found those same samples to be negative for XMRV when tested with his antiserum.

    4) Alan says mouse contamination is so abundant that another researcher found XMRV mouse contamination in different cereal boxes.

    5) Alan says it is not likely that geographical differences account for producing both positive and negative XMRV prostate studies.

    6) When asked about the XMRV antibodies found by WPI, he had no comment except to say that it must be replicated by other labs.

    7) Alan says there is mouse contamination(nucleic acid) in water that even a .2micron reverse osmosis filter cannot filter out mouse nucleic acid (contamination) from water. Thus, all the water used in the labs have mouse contamination.

    8) Alan says another researcher found XMRV mouse contamination in cereals.

    9) When asked about the antibodies to XMRV found by WPI proving infection, Alan didn’t have any comment but that it has to be reproduced by other labs.

    10) Alan says he does not know nor can he explain why patient samples have more contamination then control samples.

    11) Alan strongly says that it is a big possibility that XMRV is just a contaminant and that it doesn’t exist.

    12) Alan says if there is viruses(XMRV) in prostate tumor tissues, it is in so low a number that it doesn’t have anything to do with disease and it is just a passenger.

    Alien Rein is either 100% right or 100% wrong.

  • John

    You wouldn’t expect recent samples to ‘predate’ the 22RV1 cell line, you would expect virus in samples taken from different patients in different locations at different points in time to show more diversity than that shown by virus in a single cell line, at least that’s my understanding of the argument.

    Full disclosure- I’m still not sure about the whole XMRV thing, but if it were introduced into the human population in a vaccine scenario then it seems like all bets would be off in terms of sequence variation analysis.

  • Anonymous

    Yes, the WPI and NCI and others have found greater sequence diversity than has been published.

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