Integration of retroviral DNA into the cellular genome is essential for the production of new infectious particles. A strong argument that the novel human retrovirus XMRV is not a laboratory contaminant is the finding that viral DNA is integrated in chromosomal DNA of prostate tumors. Nucleotide sequence analyses of 14 integration sites in prostate tumor DNAs from 9 different patients previously revealed the expected viral sequences linked to human DNA. But two of these integration sites are identical to those found in a prostate tumor cell line infected with XMRV.
A search of the nucleotide sequence database with the previously identified XMRV integration site sequences revealed that 2 of the 14 sequences (from 2 patients) were identical to two XMRV integration sites in DU145 cells. This cell line was established in 1978 from the brain metastasis of a human prostate tumor. In early 2010 2007 DU145 cells were infected with XMRV, and sequences of two integration sites were determined (the database entries can be found here and here).
Identical retroviral integration sites have never been reported in independently infected cells. Furthermore, XMRV infection of DU145 cells was done in the same laboratory in which the XMRV integration sites were identified in prostate tumor DNA. The conclusion is that two of the 14 XMRV integration sites in prostate tumor DNA are likely to be the result of contamination. These prostate tumor DNA samples were probably contaminated with DNA from XMRV-infected DU145 cells.
These observations do not directly impugn the veracity of the other 12 XMRV integration sites identified in prostate tumor DNA. However, when DNA contamination occurs it is often ubiquitous. Hence the authors write:
Whilst it is conceivable that the other 12 integration sites apparently derived from prostatic tumor tissues are genuine patient-derived sequences, we suspect that some or all of them may also be the result of contamination with DNA from experimentally infected DU145 cells.
This possibility can and must be addressed experimentally.
Update: While writing this post I received an abstract from the 2011 Conference on Retroviruses and Other Opportunistic Infections (CROI) entitled “XMRV probably originated through recombination between two endogenous murine retroviruses during passage of a human prostate tumor in nude mice”. As usual I will await publication of this story in a peer-reviewed journal before discussing it further.
Garson JA, Kellam P, & Towers GJ (2011). Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection. Retrovirology, 8 (1) PMID: 21352548
Stone, K., Mickey, D., Wunderli, H., Mickey, G., & Paulson, D. (1978). Isolation of a human prostate carcinoma cell line (DU 145) International Journal of Cancer, 21 (3), 274-281 DOI: 10.1002/ijc.2910210305
Dong B, Kim S, Hong S, Das Gupta J, Malathi K, Klein EA, Ganem D, Derisi JL, Chow SA, & Silverman RH (2007). An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors. Proceedings of the National Academy of Sciences of the United States of America, 104 (5), 1655-60 PMID: 17234809
so we wait. Contaminants do not make antibodies.
They also don’t preferentially contaminate samples from patients with symptoms of illness…
Plus… since the cell line itself is of prostate tumor origin, and the samples showing integration are also prostate tissue, might this be the expected result? In other words, maybe those two integration sites that they have in common are just that, integration sites typically used by XMRV in prostate tissue.
If XMRV has something to do with the pathogenesis of prostate cancer, one would expect it to be found in human derived cell lines of prostate tumor origin AND new tissue samples from prostate tumors. Just because we didn’t know about XMRV in 1978, doesn’t mean it didn’t cause the tumor cells that were ultimately used to produce the cell line.
No because these are the exact same sites, not just similar sites. No virus has ever been found integrated into the exact same place in two separately infected cells.
Also, patient samples could well be contaminated preferentially if they are handled more, which they likely will be unless the samples are blinded from start to finish…
The attempts to discredit the work of the WPI and to even discount XMRV as a human infection are looking increasingly desperate!
The webcast of Vinay Pathak’s CROI talk is online: http://www.retroconference.org/2011/data/files/webcast_2011.htm
Scroll down to Tuesday.
No, WPI and its fanboyz are looking increasingly desperate 🙂
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Lombardi et al. and Lo et al. never used any of those cell lines. The studies were blinded, and every sample was handled in the same fashion.
I am completely lost, is CROI saying a lab procedure produced XMRV and then somehow XMRV got into humans? So XMRV is a man-made retrovirus? how did it get into humans then, maybe a vaccine ?? Im totally lost.
Nope. Just want to see our severe disease which has been ignored by the medical community for roughly 30 years have some legitimate research applied to it 🙂
I would like to ask Vincent how do you explain that Dr Mikovits submitted an abstract at CROI and was not accepted, also with the knowledge she published that paper in Science and has not presented at CROI, when others who didn’t find the virus get to present (like Switzer)
I would like to know if conflicts of interest occur when choosing presenters on a so said controversial topic such as XMRV and key scientists get left out voluntarily?
I mean, wouldn’t you like to know what Dr Mikovits has to say and give her the opportunity to answer questions?
Just candidly asking…
The study says that the prostate cancer cell line, 22RV1, was isolated by growing human tumor cells in a strain of mice called nude mice. The authors managed to find stored samples of the original tumor and isolates of each individual passage through mice as well as the cell line. What is very striking is that while the final cell line is chronically infected with XMRV, the tumor sample before it went through the mouse was not. The mice themselves harbored two endogenous retroviruses that infected the human tumor cells during their growth in the mouse. At some point these 2 viruses mixed together to form the virus we call XMRV. Therefore it is vanishingly unlikely that XMRV existed before this procedure, and it is just as unlikely that XMRV is really out there in people unless they have physically come into contact with this cell line. This and the Tower’s group studies seriously questions the validity of studies that have implicated XMRV in human disease. Moreover both studies show that mouse DNA harbors XMRV-like sequences and that these sequences are widespread contaminants of biological reagents – ie: the marketed XMRV tests and some of the hot-start polymerases used in some of the detection studies. Moreover chronic inflammatory conditions (such as CFS and arthritis) do result in the production of antibodies known to cross-react with retroviral proteins, probably due to expression of proteins from some of the person’s own endogenous retroviral loci (which constitutes about 10% of our genome – 5X more than our own genes believe it or not!). The upshot is that while there may be direct viral causes of diseases such as CFS, these new data make it very unlikely that XMRV is the culprit.
XMRV integration sites were first discovered in prostrate DNA taken directly from patients. No cell lines of any kind involved. Cell line contamination not possible there.
Section of the paper:
XMRV Integration Sites in DNA Isolated from Human Tumor-Bearing Prostatic Tissues. To confirm that XMRV can infect humans, a linker-mediated PCR method was used to map the provirus integration sites in DNA isolated from prostate tissue of men with prostate cancer and germ-line mutations in RNASEL. Cases VP234 and VP268 were selected for analysis because both were germ-line homozygous for the R462Q mutation in RNASEL and both were XMRV-positive as determined by RT-PCR performed on RNA isolated from prostate tissue (4) (data not shown). We detected XMRV provirus in DNA isolated directly from separate pieces of frozen prostate tissue from both patients. In case VP268, two proviruses were detected from the same prostate tissue sample, one integrated in chromosome 7p15.1 and the other integrated in chromosome 16q22.1 (Fig. 5 A and B). In both, integration occurred near the transcription start site of genes encoding a transcription factor. In chromosome 7, the XMRV provirus was located 2,640 bp upstream of the transcription start site of cAMP response element-binding protein 5 gene (CREB5) (21, 22), and the provirus found in chromosome 16 was located 1,816 bp downstream of the nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 3 gene (NFATc3) (23–27) transcription start site. These findings are consistent with genome-wide analysis of MLV integration sites indicating that MLV favors transcription units and integrates preferentially near the start of transcriptional units (28, 29). In DNA isolated from prostate tissue of case VP234 we identified in chromosome 17q23.2 the presence of XMRV provirus, which was 11,888 bp downstream of the transcription start site in the amyloid β precursor protein-binding protein 2 (APPBP2/PAT1/ARA67) gene (30), encoding a repressor of androgen receptor transactivation (Fig. 5 C) (31, 32).
Dong et al. PNAS
An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors
http://www.pnas.org/content/104/5/1655.full
Can it be seriously proposed that a random recombination event resulted in the creation of a novel, complete, genetically potent and infectious new retrovirus? Nature proceeds by a process of evolution and not creation. A cohort of Rhesus Macaques monkeys has been exposed to and successfully infected with XMRV. The XMRV viremia was then re-activated after 9 months, confirming the chronicity of the infection. Furthermore (and critically) anti-viral antibody titres were detected soon after infection. This response was further boosted upon reexposure. Contrary to your hypothesis about chronic inflammatory illnesses (such as CFS & arthritis), this antibody response occurs despite there being no acute or chronic inflammation in any of the Rhesus monkey tissues. Nature is complex and the idea that recombination can lead to a potent retrovirus is highly questionable.
Are we actually meant to believe that dr bob silverman created the 3rd human retrovirus xmrv, in his lab, him self ???
I dont know who did this experiment and made xmrv but I seriously doubt dr silverman did it!
WOW how did they manage it?
John.
Could you answer I question please. I do not know much about science and have difficulty understanding the journal articles.
Was a contaminated cell line used in the XMRV Rhesus Macaques study? If not, why did they monkeys become infected with XMRV if it is just a contaminate?
I’m afraid Crafter Kate that “idea that recombination can lead to a potent retrovirus [being] highly questionable” is rather undermined by the fact that it is well known to have happened before. SIVcpz (the chimp virus that became HIV-1 in man) is a recombinant of two distinct viruses that infect different African monkey species on which the Chimp preys. Furthermore a Pig Endogenous Retrovirus (PERV-A/C) is also such a recombinant. This virus efficiently infects human cells and has been a cause for concern because of the potential use of pig organs for xenotransplantation. Recombination is a mechanism of virus evolution not creation, and is entirely “natural” in infectious disease – what do you think “reassortant” flu viruses really are?
As for the antibodies, you are conflating what is going on in a monkey experimentally infected with high titre XMRV (which of course will make antibodies – if it didn’t it would be bizarre) and chronic inflammatory conditions that give rise to large amounts of cross-reacting antibody (many women with RA have been found to have antibodies that cross-react with HIV-1 yet there is no evidence of them being infected with the virus).
Mouse endogenous viruses have been tranferrred to human tumor cell lines many times by the same process that has led to XMRV infection of 22RV1 cells – you only need to look at some of them in the EM to see them.
“Gradual, accumulative recombination is a very important facilitator of natural Evolution. Recombination of two significantly disparate, partial viral sequences (as described by Vinay Pathak at CROI) to produce an infectious, potent and complete new retrovirus is Creation. Creation obviously requires specific resources targeted in a deliberate manner. It cannot be accidental. The obvious question then is… when did this occur in our oft chequered pseudo-scientific, politically-driven history?” – a Biochemical Scientist in the UK
I agree Crafter Kate.
“Gradual, accumulative recombination is a very important facilitator of natural Evolution. Recombination of two significantly disparate, partial viral sequences (as described by Vinay Pathak at CROI) to produce an infectious, potent and complete new retrovirus is Creation. Creation obviously requires specific resources targeted in a deliberate manner. It cannot be accidental. The obvious question then is… when did this occur in our oft chequered pseudo-scientific, politically-driven history?”
Someone said below, if XMRV is really out there in people, they have to physically have come into contact with this cell line.
So I ask the question if xmrv is not in mice, how have people come into contact with this cell line ?
No, both retroviruses from Pathak’s study are not that disparate, they are both MLVs. They will both cross-package each others genomes, and since retroviruses are diploid, there is potential for multiple crossing over events (recombination) during reverse transcription. It happens all the time for retroviruses and has done so over the millions of years of their history – it is a major mechanism for the generation of multi-drug resistant HIV-1 for example . SIVgsn and SIVrcm are far more diverse from each other than these 2 precursor MLVs and yet they managed to do it. The only thing that is special about XMRV is the hysteria it has generated, but this certainly isn’t creation with a large “C”, it is the evolution of a new virus through the process of recombination of two related viruses replicating in the same cell..
…which is not likely to have happened, implying that the “positive diagnoses” are due to contamination of patient samples after isolation in the lab through multiple sources. It is notable that at CROI yesterday several groups who can demonstrably rule out contamination have not been able to find XMRV or X-MLV in any clinical samples (Prostate or CFS). This includes new samples from patients with CFS previously shown by WPI to be positive. Anyone who has been told they are “XMRV+” by a PCR-based test should be very skeptical of its validity.
Some good points kate, I think I understand what your trying to say about recombination in this specific event.
I think the way ‘XMRV’ has been created is setting off many alarm bells!
Yes, of course this can be seriously proposed. In fact, it’s exactly how nature and evolution work.
This “random recombination” virus would “survive” because it “accidentally” was able to survive in its natural environment (a human cell line). The other (uncountable) random recombination events that did occur did not survive their environment. That’s the nice thing about nature: we only get to “enjoy” the random recombination events that “succeed”.
For example, what are the odds of certain species having evolved into fish in the sea? The odds are really astronomically low. Furthermore, you could argue that the odds of the evolution of fish on land are just as high as in water, when all mutations/recombinations are really random. However, when you have enough random mutations/recombinations and an environment that favors certain mutations/recombinations, fish will be “created” in the sea and not on land every time.
I had no idea we were smart enough to accidentally create the 3rd human retrovirus.
There seems to be an assumption that contamination and a wild-type virus are mutually exclusive hypotheses. A wild-type virus would be very likely to result in contamination prior to discovery. Contamination, by itself, does not distinguish competing hypotheses. Rather than plunge into the immediate debate I would like to offer a broader perspective.
Perspective: There is a certain purity and clarity to research which avoids all contact with diseased people, and there is a need for fundamental research on biology. Unfortunately, we have great uncertainty about many basic questions in biology as shown by changes in the field in the last 20 years. Our present understanding is unlikely to remain unchanged for so long.
We also have real public health problems to deal with. Many of these seem currently intractable. There is good reason to think our ignorance and that intractability are related.
Biomedical research has progressed largely through attention to specific examples. Polio might have been overrated as an epidemic, but there is no question the problems it posed led to a revolution in virology.
This brings me to questions about contamination. Assume, for the moment, there are undiscovered pathogenic viral infections, of whatever type, in the wild in humans. Assume we have no test assays to detect them at present. In this situation, researchers working with tissue samples from diseased people will have laboratory contamination. To avoid this they would have to avoid those patients. This is a good way to avoid criticism, but not good policy for investigating disease.
Prior to about 2004 there was simply no assay to detect XMRV. Stipulate there may have been such virus in the wild and the above situation would obtain.
One way to guarantee you have no contamination is to avoid finding any new pathogens. You will also likely avoid finding ways to treat pathogens.
Alexander Fleming was once shown a gleaming new laboratory. His host said, “Think of what you could have discovered with such facilities!” Fleming replied laconically, “Not penicillin.”
Agreed RRM, Nature proceeds by a process of evolution. However, this random recombination event took place in a *science lab* and resulted in the creation of a new HUMAN retrovirus, XMRV.
I’m completely furious that now this virus XMRV is in my DNA for the rest of my life and will be passed onto my future children’s DNA. A virus born in a lab which is now making me sick. Please explain to me RRM how that is evolution?
All we want are the treatments we deserve, the medications to treat this retrovirus, then we can get well and move on with the rest of our life.
It took place due to repeat passages that were performed between 1993 and 1996, the preponderance of evidence at this point is clear that it is not at all likely to have infected any humans. Also, only a virus capable of integrating into sperm or oocytes would be passed on to offspring.
Well, recombination is actually a form of evolution. From Wikipedia:
“Evolution may occur when there is variation of inherited traits within a population. The major sources of such variation are mutation, genetic recombination and gene flow.”
The assertions that you have XMRV in your DNA and that this XMRV is now making you sick are not supported by the growing body of evidence, by the way. It is much more likely that the lab that has diagnosed you as XMRV+ is wrong.
You have no idea what your talking about.
That may be, but I’d just like to point out that the type of prostate cancer being studied in possible correlation with this virus is very strongly hereditary. Also, if you listen to CFS patients, the disease appears to occur in successive generations in families as well – at least in some cases. So it wouldn’t be inconsistent.
They injected those monkeys with the virus clone they produced in the lab. The difference is that they were looking for what infection with this virus caused in those monkeys, while in the prostate cancer study they were looking for the virus in the patient samples (without the knowledge of virus presence in those samples)
There is no evidence that XMRV has caused your illness (or any other illness for that matter). Please don’t jump to conclusions from limited amount of data.
How would a virus that didn’t exist prior to 1993 be passed through successive generations?
I can’t answer your questions, as I am not part of the committee that
selected the CROI talks. I would agree that WPI should be represented
at the meeting, having begun this line of investigation with the
Science paper.
How do you know what year XMRV was mafacutured in the lab Mr Jefferys? Could be 10, 20, 30, 40, 50 years ago!?
“Analysis of 15 nude mouse strains indicated that none contained XMRV, but some strains potentially used to passage the xenograft contained both PreXMRV-1 and PreXMRV-2.”
That is from the abstract ‘Xenotropic Murine Leukemia Virus-Related Virus (XMRV) Probably Originated Through Recombination Between Two Endogenous Murine Retroviruses During in vivo Passage of a Human Prostate Cancer Xenograft’.
Potentially????
Then how can samples from the 1980’s have been found positive? How can they be finding PMRV and XMRV in the same patients. They don’t know where that cell line has been. 22Rv1 was not used in any positive study. The ME researchers have never used it. PMRV in Lo et al. has been shown to have mutated when patients were retested. The holes are too big, and research must continue.
That’s belief not science
XMRV integration sites were first discovered in prostrate DNA taken directly from patients. No cell lines of any kind involved. Cell line contamination not possible there.
Also, the study in question is unable to say where the cell line has been “Analysis of 15 nude mouse strains indicated that none contained XMRV, but some strains potentially used to passage the xenograft contained both PreXMRV-1 and PreXMRV-2.”
Identical insertion sites is a probabalistic argument and purely determined by sample size and the affinity of a retrovirus for GPG islands .
Samples in Lombardi and Lo et al were blinded and all handled the same way.
Oakes et al. and Robinson et al. both had mouse contamination. The authors of those two studies were:
Brendan Oakes1,2, Albert K Tai1, Oya Cingöz3,4, Madeleine H Henefield1, Susan Levine5, John M Coffin3,4, Brigitte T Huber1*, and Mark J Robinson1†, Otto W Erlwein1†, Steve Kaye1, Jonathan Weber1, Oya Cingoz2, Anup Patel3, Marjorie M Walker4, Wun-Jae Kim5, Mongkol Uiprasertkul6, John M Coffin2, Myra O McClure1*
Ruling out contamination is not the same as identifying MRV’s. Lo et al and Lombardi et al also rigorously ruled out contamination. Lo et al. has in fact used the unvalidated IAP assay. 0 samples were positive for contamination. The CDC has retested 24 samples from the WPI, 0 were positive for mouse contamination.
The only group at the conference that I know have retested Lombardi et al positives is the CDC. They also, as I have said, found no contamination.
It is logical to be skeptical of those claiming to have assays that are able to detect MRV’s in known clinical positives, particular when they have provided no evidence that they can.
Where is the evidence?
Finding PreXMRV-1 and PreXMRV-2 in some “strains POTENTIALLY used to passage the xenograft” is not proof.
The WPI and Ruscetti are likely to now have data on PMRV and XMRV being found in the same patients. To limit the exchange of knowledge like this is unacceptable.
Dr Mikovits was not invited last year either, nor this year. On the recordings it was recognized there was a very unbalanced panel, and someone from the audience went on to say that there was a similarity between HIV denialists and CFS patients that are X+ and chosing to be on antiretrovirals after decades of no treatments.
Johnathan Stoye said X was contamination and went on to say he got tested himself and started wondering if lab workers should be tested too… Hold on a minute… Is it contamination, or is it infectious?
Is that what science is??? it sounds more like a circus to me!!!
Funny though how several of the researchers at CROI acknowledge that the virus is replication competent whilst dismissing the risk it might pose to the human population, but are clearly paranoid that it might have infected themselves.
That’s not joined up thinking and looks to be more than a little bit hypocritical………or were they just not being entirely honest in the first place ?
So you acknowledge that the CROI was not a properly balanced representation of both sides of the debate ?
Yeah good point, and for that matter, the original Science paper never used 22rv1 either. They used LnCAP. And they say in their paper that the LnCAP line they used tested negative for XMRV and mouse contamination before they began using it as culture medium. So why are these studies focusing on 22rv1 and DU145? If they really think that XMRV positive samples are a result of XMRV (or P-MLV) already existing in the cell line before it is combined with patient samples (not the other way around), then why don’t they just test LnCAP?
And OK so what. Lets say XMRV was created unintentionally in a lab by humans. That still says nothing to whether it can cause disease in humans. Who says it can’t? Wasn’t there a study that showed MLV type viruses engineered specifically for human therapy (and were thought be be benign) actually caused cancer in the patients they were used in?
“…likely to now have new data”. Weren’t you just castigating the use of the word “potentially” GOB987? You seem to be wanting to have it both ways.
If you watch the talk you’ll see the data from the real historical samples – no conjecture, no speculation, just cold hard facts. Tumor was XMRV -ve before grafting into the mouse and became progressively more positive over several month-long passages through individual nude mice until the final cell line 22Rv1 has approx 20 integrated XMRV copies. Incidentally, they could extract a trace of mouse DNA from the tumor samples extracted from each mouse passage – ie the actual mouse in which the cell line was passeged. That DNA has preXMRV1 and 2 in it.
The word “potentially” was probably in the title because as the abstract was submitted for this meeting a few months ago, the authors would not want to make a definitive statement in the title until they were totally comfortable with the data. Now they are.
Everyone must remember however that this study is yet to be subjected to peer review and full publication. However on the basis of the data presented, it seems pretty clear cut to me that XMRV is a recombinant mERV that came from nude mice, as have many others..
No, I know they have. Labs are finding PMRV and XMRV. But everyone tends to argue about publication – guess it doesn’t matter when you want to argue that there is other evidence.
There is no cold hard fact about where XMRV has originate from. It’s a hypothesis, that is already looking extremely shaky before publication. Any mouse DNA being passed on due to grafting is to be expected. If you listen to the videocast from CROI, Pathak covers this point. They are expected to have a certain amount of mouse DNA. (1;53:20 ish)
The word “potentially” is from the Methods section by the way. It means what it means. The title of the abstract is “XMRV Probably Originated through Recombination between 2 Endogenous Murine Retroviruses during in vivo Passage of a Human Prostate Cancer Xenograft”
On the basis of the data presented, it is clear that research must continue, and those finding a greater sequence diversity must be allowed to teach others at conferences.