Authors retract paper on detection of murine leukemia virus-releated sequences in CFS patients

x or pA paper that reported finding retroviral sequences in blood from patients with chronic fatigue syndrome (CFS) has been retracted by the authors. Just four days ago the 2009 Science report of Lombardi and colleagues was editorially retracted. As 2011 comes to an end, so does the hypothesis that retroviruses are etiologic agents of CFS.

Readers of virology blog know that in 2009 Lombardi et al. published a Science report indicating they had detected the new retrovirus XMRV – first detected a few years earlier in prostate tumors – in the blood of a high proportion of patients with chronic fatigue syndrome. Many other laboratories attempted to reproduce this finding, but none were successful.

The next year Alter and colleagues reported finding retroviral sequences in the blood of a substantial number of CFS patients. No viruses were isolated in the Alter study; viral sequences were obtained by polymerase chain reaction (PCR). The viral sequences were not XMRV, but were closely related to endogenous retroviruses of mice called polytropic murine leukemia viruses. (Polytropic means the viruses can infect many species, including mice; xenotropic means that the viruses, though originating in mice, only infect non-mouse species).

The Lo-Alter finding was viewed by many (including myself) as supporting the findings of Lombardi et al., but upon closer inspection it became apparent that they only clouded the situation. The viral sequences reported in the Alter study were not XMRV, and it was not clear why CFS would be caused by such a diverse range of viruses. A second report in 2011 reported MLV-like sequences in a CFS cohort but many other studies failed to find any kind of retrovirus in the blood of CFS patients.

Earlier this year it became clear that XMRV is a laboratory-generated recombinant murine retrovirus: it arose during the passage of a prostate tumor in nude mice in the early 1990s. This finding made it highly unlikely that the virus could be associated with human disease. Lombardi and colleagues then retracted part of the 2009 Science paper that reported viral nucleic acid sequence; they noted that their samples were contaminated with XMRV plasmids. What remained of the paper were serological and virus culture experiments that were not specific for XMRV. Last week the remainder of this paper was editorially retracted by Science.

That left the Lo-Alter findings. The first warning came from the observation made by several laboratories that reagents used to carry out PCR are often contaminated with mouse DNA (an example is Singh’s study). The presence of this adventitious DNA can lead to detection of MLV-like sequences that resemble those found in the Lo-Alter study. The implication was clear: the Lo-Alter findings were wrong, a result of contamination of PCR reagents with mouse DNA.

More doubt came from a report of the Blood XMRV Scientific Working Group, which was assembled to determine if XMRV constituted a threat to the blood supply. In this study, sets of coded samples previously shown to be XMRV positive, as well as samples from healthy controls, were blinded and provided to 9 laboratories for analysis by PCR, virus culture, and serology. Two laboratories reported evidence of XMRV in the coded samples. Only the Whittemore-Peterson Institute identified positive specimens by PCR: two from negative controls, and one from a CFS patient. The Lo laboratory did not detect any positives by PCR, using the same nested assay that they had previously reported in their PNAS paper. The samples tested included 5 specimens that were positive in the Lo-Alter study.

The retraction of the Lo-Alter PNAS paper curiously begins with the assertion that the authors could not detect contaminating mouse DNA in their samples – which was most certainly present and lead to their detection of MLV-like sequences.

Although our published findings were reproducible in our laboratory and while there has been no evidence of contamination using sensitive mouse mitochondrial DNA or IAP assays or in testing coded panels…

This failure remains puzzling and unexplained; but as they report in the next paragraph, they appear to have run out of material to distribute to other laboratories for ‘independent confirmation’.

The authors provide three additional reasons why they are retracting this paper. They note that no one has been able to reproduce their findings, including the Blood XMRV Scientific Working Group. They have not been able to find (along with collaborators) anti-XMRV antibody, XMRV virions, or viral integration sites in patient samples. Finally, they mention their finding from the PNAS paper that a second set of samples taken 15 years later from the same CFS patients also were positive for MLV-like viruses. Phylogenetic analyses revealed that these sequences were clearly not descendants of the original strains. The sequence data used to make this conclusion were available for the PNAS publication, so it is not clear why this evolutionary incompatibility was not noted previously.

The authors conclude:

…in consideration of the aggregate data from our own laboratory and that of others, it is our current view that the association of murine gamma retroviruses with CFS has not withstood the test of time or of independent verification and that this association is now tenuous. Therefore, we retract the conclusions in our article.

The retraction of the Lombardi et al and Lo-Alter papers erases the published evidence suggesting involvement of a retrovirus with CFS. While it is theoretically possible that CFS has a viral origin, at the moment there are no data in support of a specific viral etiology. Some have suggested that gammaretroviruses related to XMRV might be involved in CFS. But I don’t see how a lab contaminant can point you in the direction of a bona fide etiologic agent. Contaminants cloud our vision, they do not improve it.

In light of these developments, the ongoing Lipkin study (sponsored by the National Institute of Allergy and Infectious Diseases, involving analysis of a coded panel of samples from 150 well-characterized and geographically diverse CFS patients and controls) seems less compelling. Many laboratories have failed to find any retrovirus in CFS patients, and the two papers central to this hypothesis have been retracted. Will results from one laboratory clear the matter up further? Whatever the Lipkin study finds, it will have to be validated by others – because we trust science, not scientists.

Update: The retraction has been published at PNAS.

TWiV 98: Murine musings, electric shirts, and rabid pathologists

Hosts: Vincent Racaniello, Alan Dove, and Rich Condit

On episode 98 of the podcast This Week in Virology, Vincent, Alan, and Rich review the finding of murine leukemia virus-related sequences in the blood of CFS patients and healthy donors, laboratory inventories for wild poliovirus containment, weaving high-performance viral batteries into fabric for the military, and a case of human rabies in Indiana.

Click the arrow above to play, or right-click to download TWiV #98 (58 MB .mp3, 80 minutes)

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PMRV joins XMRV as possible etiologic agent of chronic fatigue syndrome

The new human retrovirus XMRV, first detected in malignant prostate tissue, was subsequently identified in a high percentage of patients with chronic fatigue syndrome (CFS). The virus was not detected in four independent studies of CFS patients in Europe or the United States. The results of a second American study, whose publication was blocked for two months, provide support for the involvement of murine retroviruses in CFS.

The new study, a collaboration among scientists at the Food and Drug Administration, the National Institutes of Health, and Harvard Medical School, utilized samples from 37 CFS patients obtained in the mid-1990s. A key difference from earlier studies is that some repeat samples were used: four obtained two years later and frozen, and eight taken in 2010 and processed without freezing. The patients were from New England, New York State, and North Carolina and were not part of the previous study in which XMRV was detected. Control samples were obtained from 44 healthy blood donors.

Two kinds of polymerase chain reaction (PCR) assays were used to determine the presence of XMRV. In the first, DNA was extracted from PBMC to search for the presence of proviral DNA (e.g., viral DNA integrated into the host chromosome). In the second, RNA was extracted from plasma, and subjected to reverse-transcriptase PCR to detect viral RNA.

The authors found that 32 of 37 samples from CFS patients (86.5%) were positive in the PCR assay of PBMC DNA, compared with 3 of 44 samples (6.8%) from healthy controls. All four samples taken 2 years later, and 7 of the 8 samples taken 15 years later from CFS patients were also positive in this assay. The latter is an important result because it shows presence of virus in patients for long periods. Viral RNA was detected in plasma of 42% of the samples.

The authors performed additional experiments to rule out false positive results. The nucleotide sequences of all PCR products were determined to ensure that they represented the expected target sequence. Furthermore, contamination with mouse DNA was excluded by assaying for murine mitochondrial DNA – it was not found in any of the samples tested.

Perhaps the most interesting finding, aside from the detection of viral RNA in a large fraction of CFS patients, is the observation that the virus detected is not XMRV. Nucleotide sequence analysis of PCR amplified DNA revealed 96.6% nucleotide identity with XMRV, but they are clearly different viruses. The RNA genome of XMRV is a recombinant between the genomes of xenotropic* and polytropic* murine leukemia viruses (Figure). The genome of the viruses detected in the new study appears to be derived from polytropic MLVs, and could be called PMRV. This observation indicates that both xenotropic and polytropic MLV can infect humans in North America.

What is the significace of the observation that both XMRV and PMRV have been associated with CFS? Some investigators have suggested that variation of XMRV is minimal, based on the close identity of sequences obtained from prostate cancer and CFS patients. The finding of a different virus, PMRV, suggests greater genetic variation than previously thought, and could in part explain previous failures to detect MLV-related viruses in the previous four studies of CFS patients. Other explanations, including geographic differences and patient heterogeneity, are also plausible.

I find it very interesting that the authors readily detected PMRV sequences in both PBMC DNA and plasma. The authors of the 2009 Science paper have suggested that these approaches are not sufficiently sensitive to detect viral nucleic acid, and that culturing with susceptible cells must be done to amplify the virus. That explanation, which was used to explain the failure to detect XMRV in the previous four studies, is not likely to be correct. The authors of those studies might want to repeat their assays using primers more likely to detect PMRV.

Where do these findings lead next? The authors of an accompanying commentary have two appropriate suggestions:

At this juncture, it would seem reasonable to conduct extensive case-control studies in North America…using coded samples from subject with inflammatory disease to determine the frequency of MLV infection in patients with CFS. The potential transmission of MLV-related sequences from human to human should also be epidemiologically evaluated. […]it would also be appropriate to conduct interventional studies.

It is possible that these studies, which are needed to determine if MLVs cause human disease, might not have been done without a second independent confirmation of the association of MLV-related viruses with CFS.

*Xenotropic murine leukemia viruses infect non-mouse cells, while polytropic murine leukemia viruses can infect the cells of mice and other mammals.

Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, & Alter HJ (2010). Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. Proceedings of the National Academy of Sciences of the United States of America, 107 (36), 15874-9 PMID: 20798047

Raltegravir inhibits murine leukemia virus: implications for chronic fatigue syndrome?

RaltegravirThe finding that a retrovirus, XMRV, is associated with chronic fatigue syndrome has lead to the suggestion that the disease might be treated with some of the antiviral drugs used to treat AIDS. The integrase inhibitor Raltegravir has been found to block the replication of murine leukemia virus, which is highly related to XMRV. But the drug exacerbates autoimmune disease in mice which might rule out its use in treating CFS.

Retroviruses such as XMVR and HIV-1 have genomes composed of single-stranded RNA. This nucleic acid is converted to a DNA copy in infected cells by the viral enzyme reverse transcriptase. The double-stranded viral DNA is then integrated into the chromosomal DNA of the host cell, a process accomplished by an viral enzyme called integrase (illustrated).


Raltegravir (pictured above left) is an inhibitor of HIV-1 integrase that was approved for use in humans in 2007. The drug blocks the integration of viral DNA into the host genome and therefore inhibits viral replication.

The mouse retrovirus murine leukemia virus (MLV) has been linked to the development of spontaneous autoimmune disease. The mechanism by which the virus induces this disease is not known, but stimulation of innate immune responses by viral DNA might be involved.

Raltegravir also inhibits integration of MLV DNA into the murine genome. When mice with autoimmune disease were treated with raltegravir, they succumbed to autoimmune disease a month earlier than untreated animals. Mice without the disease were not affected by the antiviral drug. The authors speculate that by inhibiting viral DNA integration, raltegravir increases the amount of unintegrated viral DNA, elevating innate responses and exacerbating autoimmunity.

It’s not known if raltegravir is active against XMRV, the retrovirus associated with chronic fatigue syndrome. Given the similarity between the genomes of MLV and XMRV it seems likely that the drug will inhibit the virus. If the ability of raltegravir to treat CFS is tested in clinical trials, it will be important to carefully monitor treated patients for signs of autoimmunity. CFS has an autoimmune component which could worsen with raltegrivir treatment.

An obvious question is whether raltegrivir induces autoimmunity in AIDS patients. I’m not aware of any such reports, which is probably not surprising given the fact that HIV-1 infection leads to immunosuppression.

CFS sufferers should not despair: other antiretroviral drugs, including chain terminators such as AZT, do not allow the accumulation of unintegrated viral DNA. These compounds might be useful for treating the disease.

G.B. Beck-Engeser, D. Eilat, T. Harrer, H.-M. Jack, M. Wabl (2009). Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir Proceedings of the National Academy of Sciences : 10.1073/pnas.0908074106