Since 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
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Professor Racaniello,
Lombardi and Lo et al identified a human antibody response to a gamma retroviral infection and demonstrated that live gamma retrovirus isolated from human blood could infect human cells in culture. That can’t be explained by contamination can it?
I thought XMRV had been found intergrated into prostate cancer cells? Could that be contamination? Funny how there seems to be a bit of an Atlantic divide on this.
It’s possible that some individuals are infected with XMRV or a
closely related virus. Or that the LNCaP used for co-culture with
human PBMC were contaminated with XMRV. These are important questions
that need to be answered.
Silverman and colleagues published a paper in PLoS One in which the
integration of XMRV into the human prostate cancer cell line DU145 was
studied. They infect the cells with virus and find viral DNA
integrated into the genome of the cells. Looking at the paper, I don’t
see any reason to believe that the cells were already infected with
XMRV, although they don’t state that they checked uninfected cells.
Ah… whoops. Thanks for clearing that up.
So after all this time we don’t really have any clear evidence that XMRV is able to infect humans at all? I wonder what Silverman thinks about all this.
So how do you explain finding an immune response to the virus?
Immune response cannot be caused by a lab contamination!
Remember that WPI has found antibodies to XMRV in patient sera, and
has been able to propagate the virus in cultured cells. So there are
still issues to be clarified – see my other comment on this issue.
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Dear Prof Racaniello, I am a follower of your podcasts and blogging, I noted that in the Chicago Tribune you are quoted as saying “These four papers are probably the beginning of the end of XMRV and CFS,” although this does seem at odds with the comments in this comment section and the tone of your blog, would you be able to clarify your statement? were you mis quoted in the Chicago Tribune?
But doesn’t the demonstration of infectivity in culture by Ruscetti et al directly undermine the claim by Hue et al that the sequences encode an envelope that (as you say) “cannot attach to human cells”?
Yes, assuming that the virus obtained in culture has the same sequence
as that deposited in the database. But the WPI sequences in GenBank –
at least the full length ones – are nearly identical to the De Risi
isolate which has the MoMLV homology. There are clearly questions that
remain to be addressed.
Yes that seems reasonable.
Further research will help answer these question. Shame that the press are spinning the story as if everything is settled and XMRV (MLV- related retroviruses) is a contaminant.
The press release from UCL is ridiculous http://www.ucl.ac.uk/news/news-articles/1012/10122001
I did make that statement to the Tribune, and followed it with
comments similar to those in this blog post. I do believe that the
MoMLV homology is a big problem, but not an impossible one to explain.
I do not believe that the four Retrovirology papers prove that XMRV is
not involved in human disease.
Dear Prof Racaniello,
Dr. Yasuhiro Ikeda from the Mayo Clinic reported in a talk from March of this year that he had found some LnCap cell-lines to be infected with XMRV. Is such a cell-line capable of producing antibodies?
No, LNCap cells cannot produce antibodies.
Would it not be better if the authors of these studies were to do a replication study of Lombardi et al. and include the contamination checks that they have used to find contamination in their studies?
Dr Racaniello, many patients with Me/CFS would be eternally grateful if you interviewed Dr Judy Mikovits during one of your podcast to set things straight and even. What happened yesterday was an event of political nature, which was disseminated in the papers around the world at a viral speed (pun intended). Compare it to when the Lombardi et al. and Lo et Al.’ s papers came out, either of them did not get as much press review.
In it undeniable that science is infected with politics and enmeshed with politics. While I am no scientist, I am a patient with ME/CFS who sees the consequences of bad science, bad journalism and bad leadership (CDC) affect each and every one of us patients on a daily basis.
Please interview Dr Judy Mikovits.
I will try to have a TWiV next year with Dr. Mikovits – it’s up to her
to agree. I do agree that the speed with which the press has dismissed
XMRV based on yesterday’s reports is disheartening. I think most of
the stories were simply repeating press releases which were negative.
In the UK there was coverage in the press of Lombardi et al. There was coverage for the first negative study. Nothing since, except in the Independent online to say two studies were being held up for publication. Then yesterday these four papers were everywhere. It is always this way with ME in the UK. Largely thanks to the Science Media Centre, through which all science stories are filtered.
Here is the review process of Retrovirology
A manuscript submitted to Retrovirology will be evaluated by the Editor-in-Chief, who may decline it or assign it to an Associate Editor for review. The Associate Editor then recruits one or more of the Editorial Board to give comments, and based on these s/he will reject, accept or request revisions of the manuscript. The Associate Editor can call upon an outside expert should s/he feel the need. It is expected that the review period will not exceed three weeks. Based on the reviews, the Associate Editor will make a recommendation to the Editor-in-Chief for rejection, revision or acceptance. Rejected works are permitted one additional round of re-submission. If the resubmitted work is declined again the decision is final.
Edited by Kuan-Teh Jeang, Retrovirology is supported by an expert Editorial Board.
I’m XMRV Positive.
and if XMRV is proven to cause disease, I will one day take legal action against those that wrote the press statement, That XMRV does not cause this illness.
Mr. Racaniello. You said in the comments: “There are clearly questions that remained to be addressed”. You also said that LNCap cells cannot produce antibodies – and you probably know that the WPI found antibodies to XMRV, and it is published in the Science paper. Meaning that it just cannot be a contamination.
So why, if you think and know all the above, you told the Chicago Tribune today that “it is the beginning of the end for XMRV”?
If patient samples were contaminated at similar rates of between 67 and 85 percent and healthy control samples were contaminated at rates between 3 and 7 percent. Then how did the patient samples get contaminated more than the healthy control samples? Lombardi was a blinded study, I forget about Lo et al.
Why do you keep deleting that particular question?
hello, what’s wrong?
We have reported XMRV integration in fresh frozen prostate tissue taken directly from patients at radical prostatectomy that has never been put in tissue culture and believe this is solid evidence of authentic human infection . See Dong et al PNAS 2007 and Kim et al. J Virol 2008
That is a question for which there is no answer, but which is
speculated upon in the two Coffin manuscripts published in
Retrovirology.
We have reported XMRV integration in fresh frozen prostate tissue taken directly from patients at radical prostatectomy that has never been put in tissue culture and believe this is solid evidence of authentic human infection . See Dong et al PNAS 2007 and Kim et al. J Virol 2008
Thank you, correction appended to post.
The finding of antibodies does not necessarily disprove contamination
– there are other explanations. My statement to the Tribune yesterday
was based on my initial reading of the Retrovirology papers. However
upon further consideration it is clear that the results of all four
papers do not challenge the existence of XMRV in patients; rather they
point out potential pitfalls in doing research on this virus.
Prof Racaniello,
I’m not sure how you can claim that the 4 papers published yesterday “are probably the beginning of the end of XMRV and CFS”. On what basis do you make that claim? None of the papers yesterday were specific to CFS. Unless you believe XMRV to be purely a lab contaminant? Please explain.
And that is a cop out answer. It’s a simple question. How about a simple answer? How can a supposed contaminant choose to show up in such high numbers in ME/CFS patients and at only around 4% of healthy subjects? And it does this consistently. Surely a contaminant would show up in roughly equal numbers in both sets? Something smells rotten. So an answer then and not an avoiding of the simple question?
I’m not avoiding the question, these issues have been discussed
elsewhere and there is no need to repeat them. It’s simply not true
that a contaminant would show up with equal frequency in controls and
specimens. For example: if controls and patient samples are collected
at different times, by different individuals, they can be contaminated
at different rates. Patient samples also tend to be handled more often
than controls from a blood bank, for example. In one of the
Retrovirology papers the frequency of XMRV positives was higher in
controls than in patient samples – and all were due to contamination.
There is no simple answer until everyone checks assiduously for
contamination. That’s what these four papers are telling us.
Thanks for the updates, it is appreciated.
No matter which side an observer is on in the ongoing debate over MLV’s role in human disease, one can’t help but realize that media coverage has either a chilling, or stimulating, effect on funding and increasing /or decreasing/ scientific interest in any given area.
After today, an already difficult topic to study due to extramural funding difficulties and lack of interest (CFS and MLV’s) faces even more challenges. The other side, perhaps those who believe CFS has a non-MLV cause, will see their research as more fundable and relevant.
In today’s world, if researchers do not speak or advocate for their positions publicly, then those positions get harder to fund, collaborate with others on, and find resources to support.
Should it be this way? Maybe not. But it is.
I couldn’t help but notice a NHS story on the findings actually made praise of the media coverage part of the writeup: “The newspapers have all reported this story well, emphasising the strength of the researchers’ conclusions…”
But in the great majority of studies that returned pretty much the same levels of positives in ME/CFS patients and in the health subjects they weren’t just using PCR testing, and also they weren’t set out on trying to prove a contamination theory (and that’s all it still is), for whatever reasons and for whoever that suits. So, no, your answer doesn’t satisfy me. I find it very interesting that you and so others are so hell bent on wanting the contamination theory to be proved right to the extent that you virtually ignore those that offer up totally plausible and provable explanations that this ISN’T contamination. So, just why is that, hmm?
So, are you going to answer this very simple question too Prof. Racanello? It seems to me to be another vital question to be answered if the contamination theory is to hold any water whatsoever? Well?
While I appreciate your willingness to reconsider your initial position it makes one wonder about how much effort you apply to appropriate scientific rigor prior to making such comments, blog posts or podcasts. Your statement here does little to undo conclusions based on your previous statement.
Both sides have been eerily forthright in their views. CFS always ends up politicised like this. Claims of certainty which cannot be supported seem to be the norm. It’s why so many patients no longer trust a lot of traditional medical authorities.
I also tested positive for XMRV but this statement is ludicrous. Just because someone disagrees with the idea the XMRV-CFS connection does not mean you can sue them. Statements like this make CFS patients look like nut cases.
I found this interesting as well, especially the fact that the story hit the mainstream media the VERY DAY that they were approved.
Smith
Submission 6 Dec, Acceptance 20 Dec
Hue
Submission 2 Dec, Acceptance 20 Dec
Sato
Submission 1 Nov, Acceptance 20 Dec
Robinson
Submission 18 Nov, Acceptance 20 Dec
Oakes
Submission 1 Nov, Acceptance 20 Dec
The information is on the “Retrovirology” website
Vince, I’m not sure you realized what you stepped into:
http://wellcometrust.wordpress.com/2010/12/20/chronic-fatigue-syndrome-is-not-caused-by-xmrv/
was probably yesterday’s first headlines. The Tribune reporter who captured your initial impression isn’t exactly a friend of ours or the Lyme community seeking scientific answers to whatever we’ve developed. More important is that you were willing to take time to help us patients sort out very complex claims and information and see past some simple assertions. Thank you. See you soon with JM or at another webinar. No, I’m not paying.
If contamination is proven, a lot of XMRV+ will be suing the NCI.
If contamination is not proven, a lot of XMRV+ will be suing the CDC.
Thank you, Professor Raccaniello, for your willingness to engage in dialogue and to update some of your conclusions in the face of compelling data/arguments.
Also, because so much research is not followed up on due to lack of funding. Which of course is blocked by national health authorities.
Also, because so much research is not followed up on due to lack of funding. Which of course is blocked by national health authorities.
Are you planning to “Mia culpa” some of your comments made to the Tribune yesterday? In print and publicly? Now that you have caused possible damage for those of us who have CFS, I believe the ethical decision would be to do so.
Wasn’t it Retrovirology who was so eager to put their prestige behind the CDC findings of no XMRV? Or am I mistaken?