Tuesday, July 10, 2012

Next gen sequencing reveals that Pre-XMRV-2 is part of an evolutionarily young clade and more issues with PCR amplification of MLV-related sequences

There are a load of papers I'd love to blog about right now, but have been too busy writing grants to get them up -- this one however caught my eye today -- I think we have a lot of very interesting data yet to be revealed by next-gen sequencing - this paper, in Virus Research, compares pre-XMRV-2 sequences found in both wild-mice and inbred mouse populations.  It also seems that despite the fact that the virus appears to be a polytropic non-ecotropic virus [thus can infect other wild mice], not all wild mice in the same geo area carry the virus (in this case, Germany), and phylogenetic analysis implies that the clade is evolutionarily young and wide-spread amongst M.m. domesticus.  Also interesting is that the authors, Mayer et al., demonstrate the issues involved with PCR amplifying the gag region from these mice - and the utility of high throughput deep sequencing.  If you have access to Virus Research, you can read all about it - click here for the abstract -


  1. Thanks for posting this Dr O'Keefe!

    Great blog.

  2. Could it be that other mice are infected but again the ability to detect them is beyond science at this time?

    When you say a young clade how old would that be?


    1. sorry not to get back to you earlier; I have been working all weekend - in answer to your first question, I would have to say yes, because how can we know for sure they aren't there if we can't detect them. It really is a matter of probability however - if the correct tissues and controls are used, then chances are if it were there, it would be detected. In answer to the second question - without going back and doing some reading (sorry no time right now!)I honestly don't know. We are talking in evolutionary terms, meaning the phylogenetic "tree" analysis doesn't show a lot of evolution from the original sequences. This would be comparing the patient (ie from Lo et al.) and cell line sequences as well, that they mention in the paper. When I have done this kind of analysis on human genes, recent could be considered a few million years (its all relative). In this case, to be more specific, we might have expected that samples from different mouse strains and cell lines and humans would be more similar to other sequences from the same source - ie the preXMRV-2 sequences from humans would be more similar to each other than they are to a wild-mouse caught in Germany. And like-wise, sequences from mice of a particular strain would be more similar to each other and separate out on a phylogenetic tree. In addition, not all wild-mice have the virus, again suggesting if it were truly old, then more of them would be positive. Further sequence analysis (ie longer reads) may help to differentiate the viruses further. To caveat these comments however, I am no specialist in evolutionary virology - these are just my thoughts.

    2. There is no XMRV in humans. There is XMRV in human cell lines. And in samples from humans (because someone put it there). But no XMRV in humans. Read table 4 from the addendum.

      Please, please, please read the addendum from Ruscetti and Mikovits, read the tables, especially table 4, cross check the tables with the text and you will see what I mean.

    3. XMRV is not the viruses that were detected in ME and prostate cancer. They only share similarity in certain regions.

      You have been told before Tony that this paper shows the experiments of only two labs, not Dr Silvermans experiments.

      Patient 1199 is not in table 4. They are in table 1 and table 2.

    4. Thank you for the reply Dr O'Keefe.

      Would you therefore say that we need more data to establish the true age of these viruses?

  3. Tony Mach, with respect, no serious scientist is still suggesting synthetic (man made) XMRV is found in humans.

    The only people who were previously promoting this, are people apparently determined to stop MRV research in humans, the so-called XMRV contamination theorists who consistantly study the wrong virus in humans (VP62 XMRV) and pretend this is what was found in 'CFS', when it wasn't. This unhelpful part of science history is coming to and end, now other groups are slowly beginning to understand what has happened. Naturally, this has taken a lengthy time to occur due to all the obvious obfuscation with 'CFS' and Dr Silverman contaminating his own lab with VP62 XMRV, not the WPI's lab I might add. No XMRV VP62 ever was found at the WPI and Lombardi et al 2009 didn't solely look at 'XMRV', it found incomplete sequences of non synthetic gamma retroviral origin. Sadly, certain people pretend this never happened and concentrate on Dr Silverman's error only,for reasons that could be considered to be nefarious.

    One should see that cries of 'contamination' regarding gammaretroviruses in humans has not happened in the Prostate Cancer community. Their XMRV results still stand and no papers have been retracted. This is very important to understand.

    MRV's have been found in humans that are not VP62 XMRV in 'CFS'. This is the disturbing finding, as is the fact that consistantly the sick people with 'CFS' have much higher levels of MRV's found in their blood than healthy controls. 'CFS' patients also produce antibodies to MRV's that aren't VP62. This consistant finding by multiple groups cannot be explained away by simple laboratory contamination. Cleary, the retroviral infection found in these human groups, are pathogenic.

    Do remember that Lombardi et al (2011) found that 'CFS' patients who tested 'XMRV positive' had a distinct inflammatory profile that those healthy controls without 'CFS' did not. How curious this inflammatory profile is only found in 90%+ of people with an alleged laboratory contaminant, which of course, they never had. They had an MRV infection, a poly.

    Science needs to investigate this further. Now we know from the Mayer paper that Dr's Pathak, Paprotka & Coffin's past theories on PreXMRV-1 & PreXMRV-2 magically explaining the findings of 'XMRV' from a single recombination event, is incorrect.

    Hopefully, with haste, Science can evolve and form into something more useful for both the scientific and patient communities.

    And so we must go on to the next stage of scientific investigation. Dropping PCR and using superior methods of viral detection (deep sequencing) to aid future clinical diagnosese and, potentially, reduce the suffering and deaths of millions if and when targetted treatments can be developed.

    Certainly one would not want to put a theory of all gamma retroviral sequences detected in patients being the fingerpring of 'contamination' before science proves this.

    This would be unscientific and potentially catastrophic. Imagine the consequences for multiple disease states if science stopped now, due to a theory of preXMRV-1 & preXMRV-2 that has already been disproven.

    One would then have to question the apparent modus operandi of those who still appear determined on stopping further scientific investigation into retroviroloy. Something no ethically sound researcher or scientist, wants to happen.

    1. Anon, the Lombardi 2011 paper proves absolutely nothing because the samples were collected based entirely on a clinical diagnosis of ME/CFS, with the cytokine/chemokine profiles being analyzed prior to 'XMRV' even being discovered. The 'XMRV' status of the samples was only determined after the main body of the work was completely finished. Which is more likely- that the patient samples were contaminated at a later date vs. random clinicians located around the country diagnosing 'XMRV+' ME/CFS patients with almost 100% accuracy? Also, if XMRV or MLV or poly or whatever is so hard to find, how is it consistantly being found in such high levels in simple banked and frozen samples (ie no special collection, processing, etc. procedures)? Why all this talk of needing to test tissues and the like if it is so incredibly easy to find in stored serum?

      As for MRV's being 'consistantly' being found in more patients vs. controls, this is not what Oakes et al. found. They found that it was the healthy controls which were positive at a much higher percentage than patients were.

      And as for questioning someone's 'modus operandi', I think you mean motivation and please stop. There are rafts of papers by very experienced researchers which refute basically every single finding related to XMRV, MLV, etc.; there is no need to question anyone's 'motivations' in this regard as the most likely answer is that their 'motivation' is simply to stop what they see as valuable research dollars being wasted chasing a contaminant. These individuals could well be wrong but to constantly question their 'motivations' just makes you look stupid. Seriously, the conspiracy theories are the freaking stupidest thing I've ever heard simply because they would require the active participation by virtually every single scientist, researcher, journal publisher, academic, journalist and government official on the entire planet to be in on it, which is a ridiculously stupid suggestion and has been since its creation some time ago.

      PS- For you to say that the patients in the Lombardi 2011 paper had 'an MRV infection, a poly', goes against the title of the paper itself, which clearly states XMRV in the title.

      PPS- The chestnut about 'other scientists only looking for XMRV' is nonsense as following the publication by Alter and Lo, many many studies have also looked for broader MLV-sequences and not just for XMRV.

      Dr. Okeefe, that is a good question about the specificity of the clade being found, how would you explain the findings of Kearney et al. which found that the Huber sequences, the Lo sequences and deliberate mouse contamination were virtually identical? Also how would you explain the findings by Towers which analyzed the Lo sequences and found that there were actually sequences which were replication incompetant being found in the samples, not to mention their conclusion that the later sequences could not have come from the original sequences?

    2. Anonymous July 19, 2012 12:25 AM

      You have made several false accusations about a number of scientists because you are not in possession of the facts. You should retract them.

      The data for Lombardi et al. was submitted to Science before the cytokine profiling took place and the gammaretroviruses here are nothing to do with XMRV which is just a brand name for a sequence which has never been detected in a human being. It is far more likely that people are infected. Silverman isolated his VP62 sequence, which Coffin defined as XMRV, from three seperate patients.

      "how is it consistantly being found in such high levels in simple banked and frozen samples"

      It is not. Where are you saying this has been detected? Next generation sequencing can detect MLVs that PCR cannot detect. Even the viruses in mice are difficult to detect as described in the paper the article is about.

      Oakes et al. did not find these viruses, did not use clinically validated assays and contaminated their samples with mouse viruses.

      There is no data that refutes the positive findings. No contamination has ever been detected in the positive samples of Mikovits, Ruscetti, Lo, Alter, Singh, Hanson, Fisher and more. Different assays cannot be compared to refute a finding and they are also not clinically validating their assays in those negative studies. Those are facts and your conspiracy theories regarding the motives of those pointing them out do not wash. Also you appeals that conspiracies never happen does not hold true in a general sense. See Watergate.

      The name of the sequence that is called XMRV was altered. It is now only used to mean VP62. It does not now extend to the retroviruses detected. HIV-1 was initially recorded in a paper where the viruses was wrongly called HTLV-III. Your argument is not one to use against scientific evidence.

      Studies that claimed to have looked for other MLVs in fact used either VP62 or the virus in 22Rv1 cells. Neither is a clinical positive and the sequences are not other MLVs.

      Kearney et al. did not find evidence of deliberate mouse contamination. They had mouse cells in their lab when they ran those experiments. That is the likely source of the contamination in those new samples that they tested.

      Towers did not find the Lo samples were not capable of replication. Towers used HIV as a model for a gamma retroviruses. This is an inappropriate comparison. Their conclusions were assumptions not supported by data.

  4. Tony Mach, could you please state exactly what you are talking about regarding table 4? Also, if we agree to make an assumption that any MLV-like sequence found in humans (and I'm not referring to XMRV from cell lines etc.), but for example, the samples from the Lo et al. paper, must be somehow due to contamination (although mouse DNA was not detectable), then why do the samples keep getting contaminated with MLVs from this one, newly evolving clade? Why not with a mixture of any of the other myriad of MLVs? How can we explain that?

  5. : it is clear that the gammaretroviral sequences detected in Lombardi were nothing to do with the Vp-62 sequence amplified in error in robert Silverman,s lab. The demonstration of cell cell transmission and cell free transmission electron microscopy and positive blots for MLV proteins and the very specific positive reaction to SFFV env make the hypothesis that these results were somehow caused by contamination very unlikely.The reputation of scientists whether deserved or not is not relevent here.It is a question that so called reputable scientists have made no effort whatsoever to eliminate confounding variables in their papers and made no attempt to reproduce the methodology used by mikovits and ruscetti. For the uninitiated the results of a PCR assay are governed by a number of key variables.reagent,primer enzyme and oligonucleotide concentrations are key as are choice of primers.The concentration of magnesium and annealing temperatures determines the sensitivity of a PCR assay and its ability to detect sequence variation.The so called reputable scientists who have foolishly tried to reproduce results by changing all the independent variables which formed the basis of the lombardi experiments have done a huge disservice to science.The matter could have been settled simply enough and still could be.Retest the Lombardi samples using the same methods and test using IAP technology. The editor of science should have insisted on this simple step.He should also have insisted on experimental evidence that the antibody to SFFV env can cross react because it has never been shown to do so.Drawing fresh samples from the lombardi cohort and then trying to prove contamination by carrying out experiments in a lab full of mice viruses is the sort of behaviour that betrays the principles of science. The Lombardi samples should be directly tested for mouse contamination and if negative then the paper should be immediately reinstated.If these so called reputable scientists who have used unvalidated technology had actually behaved like competent scientists then this matter would have been resolved by now.We know that primitive methods like the ones used by Coffin pathak mcLure etc might be OK for HIV but virtually uselss for MLVs unless sample preperation is perfect. Sequence independent amplification with next generation sequencing on lymphoid tissue samples will settle the issue. If these so called reputable scientists had used home made PCR assays to diagnose HIV without having the diagnostic sensitivity of their assays established they would face long jail terms.Yet they thought that establishing the theoretical limit of detection of their PCR assays for just one genetic sequence would be OK to detect a novel gammaretrovirus that they knew nothing about.I think these scientists are like their assays ,good in theory but rubbish in practice

  6. Hi Dr O'keefe.

    I was wondering what you think of next generation sequencing. I am under the impression that it is far more sensitive then PCR could ever be at detecting a sequence present at one copy in a large mixture of DNAs, particularly for specific pathogens. Is that correct? Also is it true that it will find viruses, specifically MLV type viruses when PCR fails? Is that what Mayer et al. were showing?

    Thank you again.

    1. Mayer et al. do say this.

      "High-throughput sequencing approaches better at identifying specific viruses than low throughput methods"