Is XMRV a laboratory contaminant?

21 December 2010

XMRVSince the first observations that the human retrovirus XMRV is associated with prostate cancer and chronic fatigue syndrome (CFS), new studies have been carried out to determine the role of the virus in these diseases. The results have been conflicting: XMRV (and related retroviruses) have been found in some patients, but not in others. Whether laboratory contamination could explain the origin of XMRV has been considered by four independent research groups.

In a study of Japanese patients with prostate cancer or CFS, the investigators found that control samples were positive when examined by PCR for XMRV sequences. They traced the problem to a component of a PCR kit that contained a mouse monoclonal antibody – produced in mouse cells, it likely was contaminated with murine viral nucleic acids. This PCR kit was also used to identify polytropic murine retroviruses in the blood of CFS patients.

The results of two studies demonstrate that clinical samples that test positive for XMRV may also be contaminated with mouse nucleic acids. DNA from peripheral blood was tested for XMRV by PCR using primers specific for the viral gag gene. Samples determined to be PCR positive (19/36 healthy volunteers; 2/112 CFS patients) always contained intracisternal A particle (IAP) sequences. IAPs are endogenous retrovirus-like mobile elements, and because they are present at 1000 copies in the mouse genome, they can be readily detected by PCR. The authors conclude that positive results obtained with their XMRV gag PCR assay are due to contamination of human samples with mouse DNA.

What is the source of mouse DNA in the human samples included in these studies? Contamination might have occurred during blood collection, isolation of peripheral blood mononuclear cells (PBMC), or when DNA is prepared from PBMC. The authors note that fetal bovine serum and phosphate buffered saline, common laboratory reagents used for cell culture, appear to be involved. It is perhaps not surprising that fetal bovine serum could be contaminated with mouse DNA – after all it is known to contain bacteriophages which are acquired during slaughter of cattle.

It should be noted that none of these three previous studies prove that XMRV detected by other groups is a result of contamination. They do underscore the need for very careful analysis of PCR findings, and the inclusion of assays to ensure the absence of contamination with mouse nucleic acids.

The results of the fourth study have direct implications for the etiology of CFS and prostate cancer. These authors found that gag PCR primers previously believed to be XMRV specific can amplify viral sequences from many strains of mice. Furthermore, these primers could be used to identify XMRV in 5 different human tumor cell lines – presumably these cells had been previously contaminated with a murine retrovirus. Analysis of a human prostate cancer cell line, 22Rv1, which produces a retrovirus similar to XMRV, provided additional evidence for laboratory contamination. Previously identified XMRV from clinical specimens are recombinants between Moloney murine leukemia virus (MoMLV) and the virus from 22Rv1 cells. Furthermore, the 1182 nucleotides present in the genome of one XMRV isolate is 100% identical to Moloney virus. This sequence encodes the MoMLV envelope glycoprotein, which cannot attach to human cells, suggesting that this XMRV isolate arose as a consequence of PCR contamination.

The authors went on to compare all XMRV sequences with that of the virus from 22Rv1 cells. The results indicate that XMRV sequences from patients are interspersed with sequences derived from 22Rv1 cells. Furthermore, the virus from 22Rv1 cells is ancestral in evolutionary terms to patient-derived XMRV sequences. There is more nucleotide diversity in viral sequences from 22Rv1 cells than in all the patient XMRV sequences. The authors conclude:

Whilst our observations cannot conclusively prove that XMRV is not a human pathogen they appear consistent with the hypothesis that XMRV is not an exogenous virus transmitting among individuals. Instead, multiple lines of evidence suggest that the full length clones of XMRV originated from the 22Rv1 cell line.

How do these findings impact research on the association of XMRV with human disease? Multiple groups have identified XMRV sequences in patients with CFS and prostate cancer, and I believe that they should re-examine their specimens to determine if murine nucleic acids are present. Towers and colleagues believe this is futile; they write that “assay contamination cannot be assessed by detection of murine DNA alone since MLVs contaminate a significant proportion of non-murine”. Determining nucleotide sequences of complete viral genomes might be useful in determining the origin of XMRV sequences. An important question that has not yet been answered is to what extent XMRV and related viruses are present in the general population. Answering this question will require the use of sensitive assays that are not compromised by laboratory contamination.

Update #1: No one has demonstrated integrated XMRV DNA in the genome of freshly isolated human cells – only in cell culture. This would be important proof that XMRV can infect humans.

It should also be noted that some isolates of XMRV can replicate in cultured human cells. This observation is clearly at odds with the conclusion of one of the papers below that the presence of the MoMLV envelope glycoprotein would preclude replication in human cells.

Update #2: Two of the 6 full-length XMRV sequences identified from prostate cancer contain the 1182 nt sequence from MoMLV; the other 4 do not. Two full-length XMRV sequences isolated from CFS patients do not contain the MoMLV sequence. This explains why these viruses can replicate in human cells. The >99% sequence identity of these genomes with those of the viruses from 22Rv1 cells remains puzzling.

Update #3: XMRV integration sites have been identified in prostate tissue (study one, study two). Mea culpa.

Stephane Hue, Eleanor R Gray, Astrid Gall, Aris Katzourakis, Choon Ping Tan, Charlotte J Houldcroft, Stuart McLaren, Deenan Pillay, Andrew Futreal, Jeremy A Garson, Oliver G Pybus, Paul Kellam, & Greg J Towers (2010). Disease-associated XMRV sequences are consistent with laboratory contamination Retrovirology

Eiji Sato, Rika A Furuta, & Takayuki Miyazawa (2010). An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR Kit is amplified using standard primers for XMRV Retrovirology

Brendan Oakes, Albert K Tai, Oya Cingoz, Madeleine H Henefield, Susan Levine, John M Coffin, & Brigitte T Huber (2010). Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences Retrovirology

Mark J Robinson, Otto W Erlwein, Steve Kaye, Jonathan Weber, Oya Cingoz, Anup Patel, Marjorie M Walker, Wun-Jae Kim, Mongkol Uiprasertkul, John M Coffin, & Myra O McClure (2010). Mouse DNA contamination in human tissue tested for XMRV Retrovirology

  • Nita

    Could we CFS patients have been infected with a mouse related virus through vaccination since these finding point out that so many labs (and labs DO develop vaccines) seem to be contaminated with mouse viruses?

  • Eca01gdf

    Just because it’s difficult to know how contamination would have occured doesn’t mean it’s not a real possibility. It seems that contamination by XMRV is a common problem. But the ‘positive XMRV’ groups would have known that since Huber announced her results some time (9 months?) ago. Hopefully they would have been especially careful since then, and the two additional papers today emphasise the need for particular vigilence in the case of XMRV.

    The claims being made by the Hue group are much stronger, but it seems like others think they’re being rather presumptuous. I wonder if they’ll get the sort of criticism for this that the WPI has faced?

  • determined

    I dislike the tone of your remarks, poptone. As a sufferer of CFS for close to 30 years, I understand your frustration, but I believe that Dr. Racaniello has earned more respect than you are showing.

  • leak

    the mouse DNA,just dont understand the production in the bovine albumen

  • lancelot

    Thank you for your blog and all your great work! I have much respect for you and i hope you can help very ill patients with CFS/ME someday with your good research . However, Your tribune statement ” it is the beginning of the end for XMRV” has been very devastating to all. Do you really believe this or would you like to take it back? Thank you for your time.

  • Kelly D.

    Unfortunately you’d probably lose, unless you could prove that your individual case was caused directly by XMRV. Even Judy Mikovits hasn’t claimed causation.

  • Kelly D.

    I agree Luke. People forget that the anointed Judy Mikovits hasn’t even said that XMRV causes ME/CFS.

  • Adam

    It’s late now and Dr R is off to bed and review. PWC will be back early morning for you to fiinish the questions from tonight and more. We sure hope you take back that statement or provide a real explanation as to why you beiieve it to be true. Good nigh and Good luck! Do the righ thing!

  • cfs patient

    It is not a matter of disagreeing with the idea. When the press releases stories with headline such as “XMRV does not cause CFS” it is a logical fallacy, unscientific, and unethical.

  • Omerbasket

    So then I’d ask you to do two things:
    1) In the future, do not run and make statements like that. People’s life (Read Dr. Jason’s study about ME/CFS patients dying much sooner than healthy people) and quality of life might depend on such statements.

    2) If you now think differently – call the Chicago Tribune and tell them (ofcourse they wouldn’t make another article that says that it’s far from being the end of XMRV, and anyone who read their article would understand that they are not going to ever write it), and after they say “no”, call journalists like Mindy Kitey, Amy Dockser Marcus, Hillary Johnson and others to say that you do not think so anymore.

    And please don’t host Allen Dove in the future. We’re sick, and also sick of rude and heartless people – and even if you think Mr. Dove isn’t one of them – his statements are the statements of such a guy.

  • Carolwhitern

    I am extremely sick with CFS as well as many of my family members. I have been researching this illness for several years now and have been following XMRV / MLV research very closely and I strongly believe that this is the missing link that we have been waiting for. Please Dr Racaniello, reverse what you said about XMRV to the press. Dont buy into the political games that are going on. Rise above this! There are may of us who’s lives and family lives depend on research and hopefully real treatment for this illness in the near future! Carolyn

  • Kati

    I appreciate your response, prof Racaniello.

    the media have decided that XMRV is dead and not the cause of ME/CFS. For the community of patients, it feels like a crucifixion. However, all these people that have screamed contamination have been unable to find it. They have not bothered trying to replicate all of Dr Mikovits and al ‘s methods, reagents and techniques. All they want is to kill the truth and association with a disease that has been terribly discriminated against.

    I am inviting you to read the proceedings of the FDA Blood Products Advisory Committee. http://www.mecfsforums.com/index.php/topic,3875.30.html It starts at post 42 and transcribed by a patient herself in real time. Hopefully an official transcript will be available soon.

  • Poptone_uk

    Tough “determined”. I’ve also had ME for 30 years and your comment is patronising. Don’t you understand just how serious this situation is? Want to be ill for another 30 years? Well then, you go ahead being all nicey nicey and watch yourself get all walked over agin, and again, and agin, and agin. Times have changed. Keep up or get left behind. If you don’t like me I couldn’t give a stuff

  • Katesellers

    Hi, I’m astounded by how emotional this debate has become. Please calm down everyone!
    I’ve posted this in other places but it occurs to me that the reason everyone is so upset by the contamination papers is because an infectious virus (as XMRV was believed to be) gave people with CFS the best hope of getting medical attention and treatment as society cannot afford to ignore the threat of an epidemic. This disease has been shamefully ignored for decades and it is no wonder that patients are desperate.
    There is still plenty of evidence that CFS/ME is infectious and the search must go on for the causative organism if XMRV turns out to be a mistake.

  • Anonymous

    I think they know that. The CDC will be sued eventually anyway for the harm they have caused patients.

  • Frogsquire

    I have to disagree Kelly, the defence of “it was just science” does not permit people to commit gross abuses of human rights. Let us not forget that “scientists” claimed they have proven that ethnic minorities were inferior only 50 years ago. People need to be held responsible for the consequences of their actions, and if there is any indication that those statements have been made out of bias or false conclusions, that affect the lives of others, then the people making them should suffer penalty. There is already a crime of falsifying research data in the USA, no such crime exists in the UK. It is no wonder that the UK is the source of nearly all of these false claims.

  • Frogsquire

    I agree Poptone, the comment by “determined is highly patronising and further directed to attack you as the poster rather than answer what is a very simple question. Argumentum ad hominem has no place in a discussion.

    Everybody deserves equal respect, qualifications do not entitle people to be “respected” for false and illogical statements, such as that made by Greg Towers and Racanello. Indeed if we start believeing people merely because of the power and position they hold that leads to the greatest problems in soceity.

  • Andy Millman

    Might be an idea to do your research before you try and write articles like the above, (or give soundbites to the media). Otherwise people may think you are deliberately trying to represent yourself as an expert on a topic in which clearly you are not.

  • Dionysus

    If this was a straightforward scientific battle of ideas, then I would have no problem with the way it is being conducted. But it is not.
    The “Retrovirology blitz” on XMRV was an orchestrated attempt to squeeze the life out of
    a valid illness theory and starve legitimate research of funding, which shallow and vacuous media hacks were all too easily coaxed into publishing.

  • Dionysus

    Unfortunately, the impact of your first comment to the Tribune is not matched by any subsequent retraction or modification.

    It’s a bit like telling someone that a loved one has died and then changing the story – the damage is already done !

  • Summerflower21

    THANK GOD FOR THE CLEVELAND CLINIC!

  • rob21

    Agreed God Bless the Cleveland Clinic, Dr Eric Klien & Dr Robert Silverman.
    *3 Cheers for them*

  • Frogsquire

    “rather they point out potential pitfalls in doing research on this virus. ”

    Dr Harvey Alter, Lo, Mikovits all understand that there are pitfalls in finding XMRV. The peer reviewers of Science understand that there are pitfalls. The peer reviewers of PNAS understand this. Everyone seems to understand that there are pitfalls except it seems Retrovirology who think this non-finding demands such attention. Yet to comment on this as if it is something new is incredulous! Bridget Huber has repeatedly tried to claim contamination, both at a UK conference and at the XMRV working group. McClure has done the same; her response to the PNAS article. Those arguments were thrown out as ridiculous in open discussion, yet now those same illogical arguments are being rehashed in an absolute avalanche of papers in Retrovirology.

    Since my comment has been removed I will post it again. My question remains, why is the absolute obvious being reported by almost every UK paper and the journal Retrovirology in 5 separate papers? The true question is why such an avalanche of papers has been published? Perhaps to try to convince people that “initially” read them that XMRV is a dead issue?

    Let’s not forget that Stoyle commented on the original Science paper in 2009 stating that “it is highly unlikely that a condition as common as fatigue could be caused by one virus.” Calling ME fatigue is like calling stomach cancer an upset tummy. People so ignorant as to the nature and true incidence of this relatively rare by terrible illness have no place in the research. In fact for such people that have so traumatised people with ME with their faulty beliefs probably cannot come to terms with the abuses that they are responsible for.

    The true question is why are UK psychiatrists so against any finding of biological causes of ME. The true question is why those same psychiatrists have such control over the UK press through the Science Media Centre? On of those psychiatrists, Simon Wessely, wrote “doctors are disgusted by their ME patients.” Those are his words not mine. Simon Wessely has had desperately sick children thrown in swimming pools to force them to save themselves from drowning.

    The true debate that needs addressing is that some scientists are determined NOT to find XMRV. They are determined to derail science. Mindy Keitel raised these issues at the XMRV working group but Stoyle did not want such a discussion heard. But it is essential that such a discussion is heard!!!

    It is an undeniable fact that certain factions of the UK medical profession is hell bent on attacking this research, and unless people are aware of that fact they may on first reading, as Dr Racaniello did, believe that there is merit in something that in fact has nothing useful to add to the debate! I applaud Dr Racaniello for being open to science and having this debate.

  • Becky234532

    The UKs NHS, National Health Service, have updated there news page.

    http://www.nhs.uk/news/2010/12December/Pages/chronic-fatigue-syndrome-xmrv-virus-disputed.aspx?forumid=331851

    It mentions the fact Prostate cancer may also be down to contamination.

    “When cloning pure XMRV from the prostate cancer cells for testing purposes the researchers found that the viral DNA thought to be from XMRV was actually a mix of DNA from two different viruses. They say that this strongly suggests that contamination is the source.”

  • Rosa G

    Am not a scientist but have a suggestion … quite simply why don’t we have some medical research done on people who have received blood donations from people who have have CFS/ME to see if they have picked it up from the blood donors who have donated to them?? Apparently the unfortunate lab. experimental macaque monkeys have been show to pass XMRV from one to another by blood borne means – also, I gather it is necessary to inspect the lymphatic tissue as the virus can disappear from the blood and remain in lymph? And, by the way, lots of people who’ve had ME/CFS do donate blood!! Simple no?? We have human examples of blood borne things being transferred. Let’s look for XMRV in those who have received donations from CFS people.

  • Anonymous

    Am I being unreasonbly critical? The integration data described in Dong et al PNAS 2007 and Kim et al. J Virol 2008 are generated by PCR (yes, the dreaded word!). In-vitro recombination is also a known problem for generating PCR artefacts (for example: http://www.nature.com/icb/journal/v76/n5/full/icb199852a.html). Human genome is littered with endogenous retroviruses (7%?), some of which may share enough homology to facilitate the amplification of recombinants. I personally don’t think the data described in Dong et al or Kim et al proves XMRV integrates into prostate cancer tissue. They haven’t shown any control to show that their PCR-based sequencing method is error-proof.

  • Pingback: Back to square one? Mouse virus may have no role to play in prostate cancer and chronic fatigue syndrome after all « Lisa A. Martin()

  • Anonymous

    Dionysus is quiet correct.

    If it is one day proven that MLV-related retroviruses are not associated with ME, fine. But attempts to stop research from discovering whether it is associated, or if it is the cause, are a disgrace.

    Notice that in the UK, the BBC, the Guardian, the Independent, and the consumer NHS site (NHS choices), all have a headline stating it is not the cause of ME. What sort of journalists or medics write such clap trap with the evidence so far presented. It is an orchestrated campaign to kill this research and kill biomedical research into ME, if it were not, the headlines would read something like, ‘ME and prostate cancer may not be caused by a retrovirus.’ Even then if is pathetic to have covered those papers in lay press at this time.

  • Anonymous

    They cannot deny the association with ME without also denying the association with prostate cancer. But this research is still not able to support the claim put out in a press release by the Wellcome Trust and parroted in news outlets, that it is a contaminate. All it can do is remind people that contamination can occur. But we knew this anyway.

  • Anonymous

    There is nothing wrong with what he said and he clarifies above. These papers poke holes in the XMRV theory and do damage the XMRV-CFS link so what he said is correct.

    Racaniello is a good guy so stop the nonsense. You can’t just go attacking everyone who says anything slightly negative about XMRV or you will lose all the reputable scientist this disease desperately needs.

  • John S

    I would like to state for the record that I have CFS, am not crazy, and do not believe in suing people because they have a different opinion then I do or said something that I disagree with. There are certainly some ridiculous comments on this article and I want to assure others reading this that most CFS patients are not crazy.

    I enjoy reading what both sides have to say and the XMRV debate has intrigued me. It has brought a lot of much needed interest and research to CFS. I would be delighted if XMRV turns out to be the cause of CFS because that would mean I have some hope for treatment but don’t believe in baseless attacking of others who have different view points or want more proof that XMRV exists in people.

    I would also like to thank Dr. Racaniello for this well balanced article on the latest XMRV controversy and as others have said would love to see an interview with Judy Mikovits.

  • Pingback: XMRV and CFS – It’s not the end()

  • Omerbasket

    Anyway, I’ll just quote Dr. Harvey Atler (things he said at the last BPAC meeting that occured this month). His words says it much better than I could:
    “In those labs who do find the agent, it’s very reproducible. Year after year, same patients test positive.

    Confirmed by sequencing, reproducible over time.

    Dr. Hanson has demonstrated how critical the assays are. When they tweaked the assays, are getting findings identical to the Lo lab.

    Diversity of XMRV/MLV being confirmed in WPI lab, so XMRV is not the only agent being confirmed there now.

    Using 100s of neg controls in same lab (Lo’s), all results for contamination were negative. Lo has done what Coffin recommended to test for contamination, and results were also neg. Always neg for contam. It isn’t logical to suggest otherwise.”.
    – Dr. Harvey Alter, discoverer of Hepatitis C and the Hepatitis C virus, and contributor to the finding of the Heptitis B virus.

  • Adam

    He just clarified it with a retraction. do you see now?

  • Anonymous

    That research has already been proposed – see the latter half of the Blood Working Group webinar at http://www.youtube.com/watch?v=0HynlkG0cVc or the pdf of the slides at http://www.cfids.org/webinar/slides-121710.pdf

    It is not clear to me if the studies proposed there is actually going to be funded and performed.

  • Poptone_uk

    Fairs fair so thankyou for reconsidering your views on these papers. You have regained some respect from me at least. Please keep doing the same.

  • Margaret

    Many people have recently tested positive for XMRV. Can’t scientists collect and use their blood samples to confirm or refute the presence of XMRV in an uncontaminated manner?

  • Anonymous

    There have been instances of unplanned viral contamination of attenuated virus vaccines. Could it be cultured accidentally? Someone would have to look for it.

  • Anonymous

    For what it’s worth, in the realm of experts, there is often disagreement. The idea that things are absolutely known is often false. In a breaking area of science? Evidence goes one way, then another.

    I am certain that Dr. Raccaniello is doing his best to represent things as correctly as he can.

  • Anonymous

    You are assuming that XMRV is the cause. There are various scenarios. It could be a cause in some. It could be present in many but not the cause of any. Trying to insert certainty into an area of research that has no certainty is just not possible.

    I sincerely hope that help is found for CFS, and a cause and cure is determined.

  • Karin

    Prof. Racaniello,

    (My previous comment did not show up so I am trying to repost.)

    Since all these studies demonstrate that XMRV-like contamination is widespread in biological products, doesn’t it raise the issue of safety?

    In particular, I have been wondering if human-mouse hybrid cell lines used to produce monoclonal antibodies couldn’t be the ideal medium for the mutation of mouse retroviruses into strains capable of infecting human cells and human patients?

    This is of course pure speculation, but couldn’t this explain why XMRV strains found in patients are so close to laboratory strains?

    It seems to me that these four new studies are not necessarily implying that the Science and PNAS studies were flawed, rather on the contrary could explain some of their finding and point to a possible origin of a human-infectious mouse retrovirus and contamination routes?

  • Pingback: ‘XMRV and CFS – it’s not the end’, says virologist Vincent Rancaniello | ME Association()

  • Dionysus

    It is with this same certainty that egotistically blinkered & self-interested research groups have unreasonably dismissed legitimate (paradigm changing) biomedical research.

    Perhaps it is the innate reasonableness, with which patients have fought to redirect the focus of research, which makes them an easy push-over for these unscrupulous individuals.

    Regrettably, lazy media hacks are willing accomplices in this deception.

  • Dionysus

    As I understand it, those people who have had a +ve diagnosis by WPI / VIP dx (and others), have had it confirmed by more than one investigative technique, hence the PCR contamination issue is already irrelevant.

  • Rosgiugliano

    Thanks Prof. Racaniello for your amended statement to the press in USA (just saw it in my email) – you’re a mensch! (That is a compliment btw). What puzzles me (a layperson scientifically speaking) is, if this mousey/ratty “contaminant” stuff is in lab. equipment generally then why does not the stuff (aka XMRV/XMLRV) also show up in other searches for viral stuff of other types?? Example: if looking for say a hepatitis thingy in samples then, if the equipment is mucky with other stuff then why doesn’t all the micro-organisms show up? Sorry for silly questions (am a Arts graduate and don’t know a thing about lab. work).

  • Rosgiugliano

    Oh thanks urbantravels!! I shall go have a look at this …x

  • Sue

    Can someone explain to me why contamination keeps being thought of as a possibility when human antibodies to XMRV were found in the blood samples? Am I missing something?

  • Anonymous

    There is always that possibility with a new discovery, or with any piece of research. Other researchers are not using the methods that have been used to detect MLV-realted retroviruses, they are not able to find the retrovirus. Therefore some are saying it must be contamination, but as yet no evidence has been provided to show that is the case.

  • Murraythefrog22

    press releases like this really make me dislike the media (hopefully Alan doesn’t see this. haha). Too often it seems like they couldn’t care less about the truth, and instead just want a story that sells. I wish people could release accurate stories and consult experts for their information. *sigh* such a shame.

  • Choosepeace

    I was wondering the same thing. Along with explaining the lack of mutation, if it does, this could also go a long way to explaining outbreaks and the high incidence (both in outbreaks and sporadic cases) among those who work in education and medicine – two professions whose members are vaccinated more regularly than the general population.