The novel human retrovirus XMRV has been associated with prostate cancer and chronic fatigue syndrome. The nucleotide sequence of XMRV isolated from humans indicates that the virus is nearly identical with XMRV produced from a human prostate tumor cell line called 22Rv1. This cell line was derived by passage of human prostate tumor tissue in nude mice. Sequence analyses reveal that the genomes of these mouse strains contain two different proviral DNAs related to XMRV. These viral genomes recombined to produce XMRV that has been isolated from humans.
XMRV was originally isolated from a human prostate cancer in 2006, and subsequently associated with ME/CFS. The human cell line 22Rv1, which was established from a human prostate tumor (CWR22), produces infectious XMRV. An important question is whether XMRV was present in the original prostate tumor, or was obtained by passage through nude mice. To answer this question, DNA from various passages of the prostate tumor in nude mice (called xenografts), and the mouse strains used to passage the tumor, were analyzed for the presence of XMRV proviral DNA.
Early-passage xenografts did not contain XMRV, but mouse cells found in them did contain two related proviruses called PreXMRV-1 and PreXMRV-2. The 3€™-3211 nucleotides of PreXMRV-1, and both LTRs, are identical to XMRV save for two nucleotide differences. The genomic 5€™-half of XMRV and PreXMRV-1 differs by 9-10%. PreXMRV-1 is defective for replication due to mutations in genes encoding the gag and pol proteins. PreXMRV-2 does not contain obvious mutations that would prevent the production of infectious viruses. The gag-pro-pol and a part of the env region of this viral genome is identical to that of XMRV save for two base differences; the LTRs and the remainder of the genome differ by 6-12% from XMRV.
Comparison of the sequences of PreXMRV-1 and PreXMRV-2 indicates that recombination between the two viral genomes led to the formation of XMRV. When the sequences of PreXMRV-1 and ˆ’2 are used to construct the recombinant XMRV, the resulting virus differs by only 4 nucleotides from the consensus XMRV sequence derived from all human isolates reported to date.
The nude mice used for passage of the original prostate tumor were likely the NU/NU and Hsd strains. Neither mouse strain contains XMRV proviral DNA, but both contain PreXMRV-1 and PreXMRV-2 proviral DNA.
These data demonstrate that XMRV was not present in the original CWR22 prostate tumor, but arose by recombination of PreXMRV-1 and PreXMRV-2 between 1993-1996. When the original prostate tumor was implanted into nude mice, some of the mice harbored both pre-XMRV-1 and ˆ’2 endogenous proviruses, which recombined to form XMRV. The authors believe that XMRV originating from the CRWR22 xenografts, the22Rv1 cell line, or other related cell lines has contaminated all human samples positive for the virus. In addition, they suggest that PCR assays for XMRV may actually detect PreXMRV-1 and ˆ’2 or other endogenous viral DNA from contaminating mouse DNA.
Another possibility to explain the origin of XMRV is that it arose in mice and can infect humans. If this is true, then XMRV would have to be present in the nude mice used to passage the CWR22 human prostate tumor. No evidence for an XMRV provirus was found in 12 different nude mouse strains, including two used to passage the CWR22 tumor. Furthermore, a screen of 89 inbred and wild mice failed to reveal the presence of proviral XMRV DNA. Hence the authors conclude:
€¦that XMRV arose from a recombination event between two endogenous MLVs that took place around 1993-1996 in a nude mouse carrying the CWR22 PC xenograft, and that all of the XMRV isolates reported to date are descended from this one event.
It is possible that XMRV produced during passage of CWR22 in nude mice subsequently infected humans. Because XMRV arose between 1993-1996, this scenario could not explain cases of prostate cancer and chronic fatigue syndrome that arose prior to that date.
How can these findings be reconciled with the published evidence that sera of ME/CFS patients from the 1980s contain antibodies to XMRV? Those antibodies were not shown to be directed specifically against XMRV, and therefore cannot be used to prove that XMRV circulated in humans prior to 1993-96. Furthermore, in the absence of clear isolation of an infectious virus, antibody tests alone have proven highly unreliable for identification of new viruses.
Where do these findings leave the hypothesis that XMRV is the etiologic agent of prostate cancer and ME/CFS? All published sequences of human XMRV isolates are clearly derived by recombination of PreXMRV-1 and ˆ’2. The finding of human XMRV isolates that are not derived from PreXMRV-1 and ˆ’2 would leave a role for XMRV in human disease. As of this writing, no such XMRV isolates have been reported in the scientific literature.
Update: A second paper has also been published in Science Express today entitled “No evidence of murine-like gammaretroviruses in CFS patients previously identified as XMRV-infected”. Editors of the journal Science have asked the authors to retract their 2009 paper linking XMRV infection with chronic fatigue syndrome. The authors have refused.
T. Paprotka, K. A. Delviks-Frankenberry, O. Cingoz, A. Martinez, H.-J. Kung, C.G. Tepper, W-S Hu, M. J. Fivash, J.M. Coffin, & V.K. Pathak (2011). Recombinant origin of the retrovirus XMRV. Science Express
Vincent, is this a real replication of the Science study of 2009? Does it really imply a retractatio? or on the otherhand is it reasonable to say that is premature to retract anything and it make sense to wait for Lipkin results?
Â
This new study is not a replication of the 2009 paper, and was not
meant as such. It examines the origin of XMRV that has been isolated
from patients. I don’t want to second guess Science and their request
for a retraction, but it does make sense to wait for the Lipkin study.
If anyone is wondering why an assay that can find a low copy number of the VP-62 clone cannot find wild-type virus, the answer is relatively simple.
The chemophysical properties of the DNA are different.
High levels of oxidative stress caused by HGRV infections and commonly found in studies investigating patients with ME cause oxidative modification of nucleotide bases. The most common is the addition of a hydroxyl functional group or deamination. This (for reasons involving sterochemistry and something called twisting) means that the stacking energy and hydrogen bonding which hold complimentary nucleotide bases together are dramatically reduced. This can be compensated for by using increased levels of magnesium and lowering annealing temperatures. Another strategy is to use an RNA template to begin with. MLV viruses can’t replicate in resting cells so XMRV will exist in such cells as preintegrative complexes and integrated highly methylated proviruses. ROS oxidation induces a pattern of base substitution or inversion in the provirus affecting primer annealing. The presence of certain oxidised bases or even abasic sequences can stop taq polymerase “in its tracks†leading to an absence of product. Unless PCR reagent concentrations and cycling conditions are adjusted accordingly the virus will escape detection. Slverman was able to adjust his assays to find gag sequences which were already present and was fortunate enough to isolate XMRV from RNA hence largely avoiding these issues.
The effect of activating PMBCs would be to allow the integration of “fresh†proviruses as the preintegrative complexes are nowhere near as prone to oxidative damage. This would also allow the creation of viral RNA.â€
This study wasn’t a “replication” of Lombardi et al., but the paper by Knox et al., also released today and also freely accessible on the Science site, is. Specifically, Knox et al. retested 61 patients from Incline Village, NV, 43 of whom had tested XMRV-positive by VIPDx/WPI’s testing service. The archived clinical specimens from those patients, and more recent samples from an overlapping cohort, all tested negative for XMRV by PCR, antibody, and cell culture. Some of the patients had even had duplicate samples taken on the same day by the same phlebotomist, with one sample sent to WPI/VIPDx and the other to the Knox et al. lab.
I can’t speak for Science’s editors, but it seems pretty clear that the combination of these two papers, and the ten or so earlier ones that have also seriously undermined the XMRV-causes-anything hypothesis, led them to ask Mikovits to retract the earlier paper. When she refused, they issued the Editorial Expression of Concern. As for the Lipkin paper, it’s irrelevant whether it’s redundant – the budget was allocated, the project is underway, and results are due by the end of the year. It will be done. It does seem like these other studies have stolen his thunder, though.
On a separate note, Vincent, you should probably put comments on moderation if you haven’t already. Past discussions of XMRV on this blog have often been overwhelmed by the Dunning-Kruger effect.
Dr. Racaniello – How common is it for a journal to ask scientists to retract a paper? In the case of fraud such as Wakefield’s paper it would seem obvious to ask for a retraction although the Lancet did not, but other issues seem less clear cut.Â
Is there a standard policy? What factors are taken into account?
Sending patient samples to VipDx that were negative according to Levy, doesn’t reallly say anyhting that we didn’t already know.
If I were Levy, I would have sent samples of HEALTY controls to VipDx (telling them this were patient samples). VipDx finding 50+% in those samples would have really sealed the deal N-ray style.
But I guess the above is just some more Dunning-Kruger effect in action… 😉
Dunning-Kruger effect on an educational blog? Â *blink*
Alan, you’ve made previous comments showing that you have very little familiarity with the biomedical research in this particular disease, but I wouldn’t go so far as to say you are an unskilled person making poor decisions, reaching erroneous conclusions and too incompetent to have the metacognitive ability to appreciate your errors just because of ignorance – no matter how tempting. Pigheadedness is a human trait not limited to scientists, politicians or the lay public.  If comments bother you or conflict with your own confirmation bias you can always  just skip past them without denigrating the person (s) posting their opinion.Â
What these studies show is that XMRV did not originate in wild mice but was created in the lab through recombination of two common endogenous viruses found in lab mice. What is also clear from all the press around XMRV, is that contamination of biologiocal products is very common.
OK fine. Then what? Does this say anything about XMRV causing or not causing disease in humans? NO.
The fact that XMRV does infect human cell lines, and is ominpresent in biological products, leaves the door wide-open to the possibility that humans get contaminated with XMRV by injected biologicals (e.g. vaccines, monoclonal antibody treatments, etc…).
Discarding the possibility of XMRV causing disease in human just based on the fact that XMRV was created in the lab is very bizarre.
Quite the opposite, the facts above should drive everyone to look for a possible role of XMRV in human diseases, but NOT by looking for it in blood, since it is now pretty clear that XMRV is next to impossible to find there. Why not look for it in other cancerous tissues, gut biopsies, etc…?
Concerning timing (prostate cancer and CFS cases originating before the 1990’s), how about the idea that there might be different origins for a disease? That does not mean that XMRV does not cause similar symptoms or is not associated with variants of a disease.
Then last but not least: XMRV was shown to be a recombinant of two endogenous retroviruses present in common lab mice. Why couldn’t the same event have happened before in other cell lines or during other biological experiments? Would this be very unlikely or not so much?
Pingback: De to nye negative XMRV-studiene publisert i Science i dag « Rutts tankespinn og ME-nyheter
I don’t have any hard numbers on how often journals ask scientists to
retract a paper, only my sense that it is very rare. Author
retractions are more frequent, but I’m only aware of a handful of
those in a year. I can recall a few other cases in which the authors
were asked to retract a paper, but in those cases scientific fraud was
involved. That’s not the case here. I don’t believe there is a
standard policy that enumerates how such decisions are made; if they
exist they are not public.
The fact remains that all human isolates of XMRV described to date
originated from the recombinant virus produced in laboratory mice in
the 1990s. Whether or not other related murine retroviruses could
cause human disease is certainly a valid question, but there is little
evidence to support such associations or research into identifying
them.
i don’t know if this is your sense of humor…if so, it does not come across well via internet; or you are just an ass.
people are really really sick…they have lost their lives due to this disease (whatever the hell you want to call it)…when you denigrate people who are passionately fighting for their lives with such comments as the one above you reveal what a compassion-less twit you are.
perhaps suggesting VR moderate to cruel, offensive postings would be called for….but to call a group of sick ppl…delusional and stupid, really takes the cake.
i really hope you or someone you love never gets this disease because you will greatly regret the stmts you have made to and abt sick patients over and over again.
if you were making a joke, forgive me…but it WAS not funny.
i’m surprised VR let’s you denigrate the value of his blog with your snide comments to ill people.
For good incite into the practice of retractions and papers that get retracted.. there is a great website called  -  retraction watch – not sure of this site’s policy on supplying links..so I’m not going to try…
I don’t see anything offensive in that comment. Could you show me what made you attack Alan Dove?
As a perfectly good ‘wild type’ example of this Dunning-Kruger effect, Gerwyn just asked himself who the hell this Bruces Alberts is and if he “sought expert biochemist opinon” before writing his editorial:
http://www.mecfsforums.com/index.php/topic,7600.msg91526.html#msg91526
It’s at http://retractionwatch.wordpress.com/. Links are welcome here.
Hi Drosha,Â
I can’t speak for others, but my paraphrase of the Dunning-Kruger effect  in my reply to Alan starting with the word unskilled pretty much speaks for itself as to how offensive the remark was.Â
Most people don’t take it well when you call them among other things too incompetent to have the metacognitive ability to appreciate their errors. Â
While he may disagree with posters there are more tactful ways to disagree with people – a lack of formal training in science and being stubborn rarely translate into the Dunning-Kruger effect. Â
I wonder at what point the WPI will be able to admit there lab is contaminated or if they will be able to admit they are wrong if they discover contamination themselves. Will it take all the coauthors of the science paper to back out? Will it take them not finding XMRV in the blood working group? At what point is enough, enough? They seem so emotionally and financially invested in the XMRV CFS link
that they would not be able to walk away from it even if they are wrong. Also admitting they are wrong at this point would almost certainly be admitting they are incompetent. There have been so many red flags that have been raised and they have basically responded with, no we can not possibly be wrong, everyone else must be wrong.
As a CFS patient this whole thing is tremendiously frustrating. The science paper was great to bring attention to CFS and brought a great deal of hope to CFS patients who have seen very little research on CFS in over 20 years. I am afriad the WPI venturing more and more into the extremes damages CFS image in the research world and makes researchers not want to touch it. So many resources have been used on XMRV that it is sad to think how far the understanding of CFS could be pushed forward if these resources were used to understand the biological dysfunction in CFS patients. I still have hope researches will continue to pursue other avenues of research on CFS but I fear that we are going to see a reversal to little or no research that we have seen in the pre-XMRV CFS link years as XMRV is proven to not play a role in CFS.
First of all this is not only an educational blog but also a scientific blog (mostly scientific). Secondly I’ve seen several people in this blog comment boards who reach erroneous conclusions even though they have limited scientific skills. Saying somebody is unskilled in science is not (and shouldn’t be) offensive if that person indeed doesn’t have the scientific skills or the education. It may sound offensive to those who are unaware of their skill level.
As Benjamin Franklin once said “being ignorant is not so much a shame, as being unwilling to learn”. I don’t think Alan is referring to those who are willing to learn and ask questions, but to those who have a strong cognitive bias and are unwilling to accept the scientific method (which shows that they are not skilled in this specific part of science).
For the record. Do you think papers on XMRV and prostate cancer should also be retracted?
The Dunning–Kruger effect is a cognitive bias in which unskilled people make poor decisions and reach erroneous conclusions, but their incompetence denies them the metacognitive ability to appreciate their mistakes.[1] The unskilled therefore suffer from illusory superiority,
rating their ability as above average, much higher than it actually is,
while the highly skilled underrate their own abilities, suffering from
illusory inferiority. Actual competence may weaken self-confidence, as
competent individuals may falsely assume that others have an equivalent
understanding. As Kruger and Dunning conclude, “the miscalibration of
the incompetent stems from an error about the self, whereas the
miscalibration of the highly competent stems from an error about others”
_______________________________________________________________________
sorry, as a very sick patient who is hoping that scientist and doctors find away to treat me/cfs (xmrv related or not); i find this comment highly insulting. i am not a scientist. i come here because VR breaks down scientific theory in a way that lay ppl can understand.
anytime AD has spoken abt me/cfs he has been condescending and flippant…we know his wife believes that the the disease is a psycho-somatic disorder…most of us who are ill with this disease believe differently…we experience it every day….we for the most part were healthy, lively active ppl before being struck down…not depressed malingerers searching for attention, ppl who were abused as children, or ppl looking for a way out of working.
dosha, it is clear you are a big AD fan….great….i am not…i find him offensive and condescending. let’s just agree to disagree. my message was to him not you…can he not speak for himself…or am i too ignorant for him to respond to.
if VR does not want non-scientists reading his blog because we are too ignorant, unskilled, delusional, etc. then he should change it to a member only blog for scientist.Â
that’s all i have to say on this subject.
Thanks for your continued commentary on this. All very interesting.
Everyone is welcome here. There are bound to be disagreements, which
is not a problem, unless they disintegrate into brawls. There are good
scientific questions here that should not be obscured.
1. Just because I agreed with Alan doesn’t mean that I’m his fan.
2. I just asked the reason why you wrote that very angry reply to Alan, despite the fact that he didn’t specifically refer to you when he said The Dunning–Kruger effect. This is an open forum, if you fail to acknowledge that maybe you shouldn’t be here.
3. People can certainly be unskilled on a subject but willing to learn and not make poor decisions or acknowledge their mistakes. I’m pretty sure I’m unskilled in a lot of different subjects, as long as you’re willing to learn, there is no problem (which I specifically pointed out in the last paragraph of my last comment which you clearly ignored)
4. Your last paragraph is a clear overreaction to something that is not specifically directed at you and a clear misjudgement of Dr Racaniello’s attitude towards this subject.
The study by Levy, Knox et. al, while interesting, is not a replication attempt at the 2009 Lombardi paper. Â Because of this it fails to clarify anything. Â In fact it confuses the situation more. It adds new variables instead of reducing them. Â After all the previous failed attempts at looking for this virus with new methodologies, Levy should have retreated to the original 2009 study methods to see if they could be independently duplicated and produce the same results. Â It is really inexcusable at this point to do anything but an exact replication. Â I fail to see how this paper is worthy of publication in Science, and with a retraction request at that. Â It doesn’t answer any of the outstanding questions about XMRV and its association with CFS generated by the original study. Â Its just another data point using an novel line of inquiry. Â
Also, the Levi paper openly suggests that the discordant results are due to “contamination of reagents or cell line”, but it doesn’t directly address that possibility in its experiments, making that statement speculative at best. Â To show the positive results are due to contamination, one must design a study to find the source of the contamination and show it in an experiment. Â In doing so, such a study would have to answer why, if contaminated reagents or cell lines are the cause of (false) positive results, do only the samples from sick people with CFS test positive, despite being blinded? Â Why then would the water controls or patient controls not also test positive at the same rate?Â
WPI and Mikovitz were biased from the very beginning, driven by a funding source which valued an easy answer over the truth. As Mikovitz and her gang of morons continually bury their heads deeper in the sand, the field will move on without them. It’s been enraging to watch even this blog continually kick the can down the road, as study after study made it absolutely plain that the CFS-XMRV connection doesn’t pass the laugh test. Alas, there was always a “well maybe…” Hopefully this can finally lay this corpse to rest; it’s been rotting for a while now.
I actually feel sorry for you and the rest of the patients suffering from CFS. Any increased exposure CFS has gained in the national media spotlight within the context of poor science, misinformation, and blatant conflicts of interest will ultimately be to the detriment of any patient who wants an effective understanding of CFS pathophysiology with treatment and cure options to follow.
‘While studies now cumulatively and convincingly exclude a role for XMRV
in prostate cancer and CFS, they do not rule out the possibility that
some other virus or viruses might play a role in either disease. It is
known that a variety of viruses contribute to about 15 percent of known
human cancers.’Â Update 31 May 2011.
http://www.cancer.gov/newscenter/qa/2011/xmrv_qa?forumid=331851
I’m not quite sure about your last sentence. CFS received very little to none attention before so there isn’t really much which can be taken away. Maybe some scientists feel encouraged now, to solve the CFS puzzle or at least to take a closer look at it.
According to Annette Whittemore, this finding is “not relevant to the Lombardi study.” http://files.me.com/jdj88/tnmi0f
Perhaps if someone were able to put together a coherent contamination arguement they would take it seriously. At the present time it laughable to consider any such hypothesis exists.
The sequence analysis is far from laughable. It is quite clear that
all the human XMRV isolates described to date are all derived from the
XMRV recombinant produced in mice in the 1990s. To think that such
data are laughable is not an option.
It is not possible to draw such a conclusion from the evidence in Paprotka et al. Even if there was sufficient evidence to show the virus was create during xenografting. Which the paper is also not able to prove, nor provide solid evidence for.Â
The authors have failed to look for PreXMRV-1 and 2 in wild type mice. XMRV could just as easily be ancestral to what they call preXMRV-1 and 2.Â
The same issues from CROI are also still relevant. They do not know if the mice containing PreXMRV-1 and 2 were used in the creation of the cell lines and are not able to state that their assays could detect low titire virus in the earlier generations, which were not treated with testosterone.Â
The cell line has also never been in the WPIs lab and thus cannot be the source. It also says nothing about virus infecting humans.
You are either missing the point or choose not to acknowledge it.
There is zero question that the recombinant XMRV virus produced in
mice is the virus that has been isolated from CFS patients. Your
arguments, specious as usual, do not negate this fact.
It does prove that Levy’s assays are not capable of detecting known positives.
That is an assumption, not supported by evidence.  See my post above.
The same virus in some patients, not contamination. The arguement put forth by Paprotka also does not account for the other variants. Some of which the WPI added to the GenBank only the other week.Â
Knox et al cannot be described as replication study. The conditions are not those from Lombardi et al. The paper also references Lo et al.  These assays were also not validated.
It is therefore unscientific to claim, without such evidence, that any of the positive studies have been undermined.Â
Again, you are suggesting that Lipkin would waste money. Lipkin has come out in support of continued research.
“Calls to retract the paper at this point are premature,†“Calls to retract the paper at this point are premature,â€Â “We need to let this study take its course, look at the data in a coherent fashion and figure out what it tells us.†While interesting, “the publications don’t dissuade us from continuing our work.†http://www.bloomberg.com/news/2011-05-31/chronic-fatigue-study-that-sparked-ban-may-have-been-flawed.html?forumid=331851
But it should be the default assumption, otherwise you would rapidly end up saying “everything causes everything.” We should be trying to prove that XMRV causes human disease, not assume that it does then ask for proof that it does not. This argument is reminiscent of MMR & autism. “You can’t prove it’s not therefore perhaps it is….”
There should be no default assumption at all. Â We should be trying to test the hypothesis laid out in Lombardi et al., Urisman et al., Lo et al., etc. Â That would require replication of their methodology, not the replication of assays never demonstrated to be capable of detecting the virus. Â This argument is reminiscent of HIV.
Pingback: Our Cross to Bear | Alan Dove, Ph.D.
Yes.
Pingback: XMRV: Trials and Tribulations | Research1st
I think the argument is more reminiscent of Bozo the Clown.
If there is any known positive, it is you:Â
No matter what these new studies show, we know you remain “positive” about a  possible XMRV/CFS connection!
Is that the only data you are presenting as an argument?
Your English is a little muddled there. Â Know positives are those that have been found with a validated assay. Â This can be achieved by using multiple assays and through retesting by another lab. Â Lombardi et al did validate their assays. Â Â
probably the one most relevant to this discussion was the forced retraction from the Lancet of Andrew Wakefield’s study that suggested a link between MMR vaccine, autism and crohn’s disease. Another study that caused a great deal of distrust between patients an doctors and required a huge amount of research time and money to fully disprove it (and a subsequent investigation that has revealed serious ethical concerns). The damage this has done and is still doing in terms of uptake of vaccination incredibly serious.
Could this recombination ever have happened in wild mice?
I’m still concerned.  You are speculating on the origin of XMRV.  No scientific evidence has been presented that mice were in involved in creating that human retrovirus.  The same goes for Coffins  assays being clinically sensitive enough for a low copy number virus.
This is nothing like HIV. Minor changes in the PCR methods such as those in the XMRV literature do not convert 60 to 70% positives into all negative in the case of HIV. There are many HIV assays which vary much more than the PCR assays that detect XMRV. If you can only detect a potential pathogen with one brand of Taq polymerase, for example, that makes it very suspicious that it’s not a real finding.
As for “validated assays” – there are no universally agreed clinical positives, and there aren’t ANY validated assays. Your arguments are all based on the assumption that the WPI findings are correct and sacrosanct, and you seem unable to accept that there are two possibilities here, one that XMRV is a real human pathogen, the other that it is a lab contaminant. Your arguments are entirely circular and begin with insisting on replicating something that may not be true. It does not seem to have occurred to you that a reason for failure of replication is that the Mikovits lab was simply wrong (the alternative being that everyone else is wrong). I am afraid that it is really clinging to straws to say the PCR methods weren’t *exactly* the same when in other labs viral culture and antibody assays have also failed.
This is a big disappointment to everyone following this field, don’t get me wrong. There is nothing that we all want more than a fix to this illness. It is beginning to look like this isn’t it. Nevertheless, if there is an about-turn and we can prove this virus IS the cause of CFS I for one will be delighted. Remember this isn’t about taking sides, it’s about finding the truth to help those who are suffering – and we can’t find only the truth that we want to find. Unfortunately I feel you want this a bit too much and this has blunted your objectivity.