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

  • Denlander

    The story becomes more confusing as time passes. If the positive tests for the XMRV / MLV are due to contamination, how do we explain the original negative tests, negative for controls and test subjects. Are the test reagents coming from different sources or is the contmination local to the test lab.
    Having accepted that there may be contamination in the PCR test, this does not explain positive antibody response in serology tests.
    Derek Enlander MD
    New York

  • lancelot

    These british bastards can’t do anything right! all their studies are all biased to prove their psychological model of CFS. UK=Rubbish = Genocide

  • sb6

    I think I agree, for example in the Kim et al. paper they used 80 cycles on the prostate cancer tissue samples, surely this is an unnecessarily high number?

  • Lionel Berthoux English

    Hi everybody, just passing by.
    The XMRV debate is pretty alive around here (for lack of a better understatement).
    I am a retrovirologist but I am not involved at all in XMRV research. I feel for my colleagues who have produced the now famously negative data, though, and are now the subject of angry comments here and elsewhere. A few thoughts:
    - It is nonsense to believe that scientists would somehow have an interest in getting negative data, or somehow be under pressure to not link prostate cancer or CFS to a new virus. It would be in the interest of everybody (patients, scientists and pharma alike) for such a link to be established, especially since we already have molecules at our disposal to target these viruses.
    - *Trust me* on that, it is far more exciting to get positive data rather than negative ones. Virtually all cases of scientific misconduct involve scientists who have manipulated data in order to publish falsely positive results. Grant money, career advancement and visibility come with discoveries, which is just another name for “positive results”.
    - A revealing comment I read here was: “They don’t care whether XMRV causes CFS because they don’t think CFS is a real disease”. I don’t think that any of the retrovirologists involved have any strong opinion on the medical aspects of CFS. Frankly, I would bet that most of them had only a vague knowledge of the disease until the first paper came out. You should take that as additional evidence of impartiality.
    - Although being angry is quite understandable when one is sick with a disease of unknown origin, what good does it do to get a wrong answer on the causative agent? What purpose would it serve to direct a big chunk of CFS research money into testing antiretroviral drugs in patients, only to discover 5 years later that retroviruses do not cause CFS after all? Although the field might be moving slowly from an outside perspective, I am personally astonished at how fast the studies are coming out. A lot of people are working hard to get answers and to get them quickly.
    - Finally, I’d like to give credit to the people behind this blog for allowing many of us to publish comments here, even when they are strongly opinionated and nonscientific in nature (which applies to this post as well).

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