Dr Racaniello on XMRV/Contamination/Retraction

Jemal

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There is a new post about XMRV possibly being a laboratory contaminant on Vincent Racaniello's virology blog:

http://www.virology.ws/2010/12/21/is-xmrv-a-laboratory-contaminant/

The Chicago Tribune is also quoting the professor in an article about the possibility of contamination:

These four papers are probably the beginning of the end of XMRV and CFS," virologist Vincent Racaniello of Columbia University wrote in an email. "They don't prove that XMRV (and related viruses) don't cause CFS, but they go a long way to explaining many of the different findings in different labs."
http://www.chicagotribune.com/health/ct-met-chronic-fatigue-xmrv-20101220,0,5526992.story
 

Cort

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Here's Racaniello's complete blog

http://www.virology.ws/2010/12/21/is-xmrv-a-laboratory-contaminant/

Is XMRV a laboratory contaminant?

21 DECEMBER 2010

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.

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
 

CBS

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Since his original post, Dr. Racaniello has added the following three updates:

Update #1: [The following was either deleted or I assume, taken from one of the five articles and Racaniello is now questioning this statement. It was posted in "strikeout" font (not available on PR forums) which is a font used when the author wants to show that something they wrote has been deleted. I'm substituting the strikeout with the color red]

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.
 

Cort

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Raccaniello has definitely reconsidered his comments to the Tribune author. He is profvrr

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?

profvrr
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.

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"?

profvrr [Moderator]
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.

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?

profvrr [Moderator]
No, LNCap cells cannot produce antibodies.

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?

profvrr [Moderator]
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.

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.

profvrr

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.
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Cleveland Clinic - Eric Klein, Cleveland Clinic [Moderator] 2 hours ago in reply to profvrr
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

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.

profvrr [Moderator]
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 t
 

urbantravels

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I've never been able to post to the Virology blog comments section, and now someone I know is trying to leave a comment and it won't work. He tried creating a "Disqus" account and it doesn't seem to want to go through. I know some of you here have posted comments to TWIV, what's the secret to making it work?

ETA: is there merely a delay for moderation? I've just tried to leave a comment myself and it simply seemed to disappear when I hit the "post" button.
 

lancelot

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More posts of Prof R poorly defending contamination :


Gob987 [Moderator] 2 hours ago
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.
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profvrr [Moderator] 2 hours ago in reply to Gob987
That is a question for which there is no answer, but which is
speculated upon in the two Coffin manuscripts published in
Retrovirology.
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Poptone_uk [Moderator] 1 hour ago in reply to profvrr
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?
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profvrr [Moderator] 1 hour ago in reply to Poptone_uk
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.
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Poptone_uk [Moderator] 1 hour ago in reply to profvrr
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?
 

urbantravels

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Those of you who have been successfully able to post comments on the Virology.ws blog - were you using Mac or Windows? Which browser?


And just in case I'm going nuts - the "disqus" thingie tells me that I have made one post and received one "like", but I don't see the post! Does anyone else see a post by "urbantravels" on that blog?
 

Jemal

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And just in case I'm going nuts - the "disqus" thingie tells me that I have made one post and received one "like", but I don't see the post! Does anyone else see a post by "urbantravels" on that blog?
I can't find your comment, sorry.
 

LaurelW

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Great! Now do you want to take bets on whether Ms. Tsouderos will actually print the retractions? :tongue:
 

Jemal

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Great! Now do you want to take bets on whether Ms. Tsouderos will actually print the retractions? :tongue:
Ha, I know what I am betting. Very glad he made this retraction and not only because it helps our cause.
 

lancelot

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He is obviously a man of high integrity, high morals, and so humble as to be able to admit a mistake and take it back.

You surely won't see this from british bastards(Weasel, McClure, Towers, etc)
 

Jemal

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He is even writing a letter to the Chicago Tribune, to retract his statement.

I asked Ms. Tsouderos and she said the best way to do this is via a letter to the editor - which I am writing now.