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TWiV 164: Six steps forward, four steps back

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xmrvHosts: Vincent Racaniello, Rich Condit, and Alan Dove

Vincent, Alan, and Rich review ten compelling virology stories of 2011.

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Ten virology stories of 2011:

  1. XMRV, CFS, and prostate cancer (TWiV 119, 123, 136, 150)
  2. Influenza H5N1, ferrets, and the NSABB (TWiV 159)
  3. The Panic Virus (TWiV 117)
  4. Polio eradication (TWiV 127, 149)
  5. Viral oncotherapy (TWiV 124, 131, 142, 156)
  6. Hepatitis C virus (TWiV 130, 137, 141)
  7. Zinc finger nuclease and HIV therapy (TWiV 144)
  8. Bacteria help viruses (TWiV 154)
  9. Human papillomaviruses (TWiV 126)
  10. Combating dengue with Wolbachia (TWiV 115, 147)

Links for this episode:

Weekly Science Picks

Rich – Fundamentals of Molecular Virology by Nicholas H. Acheson
Alan – Fetch, with Ruff Ruffman
Vincent – Year end reviews at Rule of 6ix and Contagions

Listener Pick of the Week

Garren – Trillion-frame-per-second video
Judi – iBioMagazine
Ricardo – Brain Picking’s 11 best science books of 2011

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

This year in virology

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XMRVFor some time I have thought about reviewing this year’s topics on virology blog in 2001, not only to get a sense of what I thought was significant, but more importantly, to highlight areas that need more coverage. I went through all the articles I wrote in 2011, put them in subject categories, and listed them by number of articles. The results are both obvious and surprising.

I wrote most frequently about the retrovirus XMRV and its possible role in chronic fatigue syndrome and prostate cancer. This extensive coverage was warranted because we had an opportunity to learn how disease etiology is established, followed by development of therapeutics. By the end of the year we learned that XMRV does not cause human disease, but the journey to that point was highly instructive.

The next most frequently visited topic on virology blog was influenza. Writing often about this virus makes sense because it is a common human infection that occurs every year, and controlling it is a continuing goal of virology research.

There were five  posts noting the death of virologists, colleagues, or someone I thought made a substantial impact on my career.

I wrote more about poliovirus than any other virus except XMRV and influenza. Eradication of poliomyelitis continues to be difficult and faces periodic setbacks.

I only wrote three articles about topics in basic virology.

Like many others, I find the biggest viruses and their virophages compelling.

The past year saw the release of Contagion, a movie about a virus outbreak. Look for an analysis on TWiV in 2012.

The state of science education and science funding is becoming more of a concern. It is not a topic I write about often – I prefer to focus on the science of virology – but for future scientists it is extremely important.

The other posts covered a variety of topics and viruses, including HIV, human papilloma viruses, hepatitis C virus, and smallpox virus.

What have I learned from looking back? The best covered viruses – XMRV, influenza, and poliovirus – deserve the attention. I am surprised that there were so few articles on important viruses such as HIV, HCV, rotaviruses, and herpesviruses. That shortcoming will have to change. I did not write enough about basic virology. One could argue that teaching a virology course is enough – but I think that concise, informative articles on basic virology are very useful. I’ll try to do more of that in 2012. There is one topic I’d like to write less about, but over which I have little control – the passing of scientists.

Thank you for coming here to learn about virology.

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

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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.

Science retracts paper on detection of XMRV in CFS patients

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Bruce Alberts, Editor-in-Chief of Science magazine, writes that the journal is retracting the 2009 paper describing the detection of the retrovirus XMRV in patients with chronic fatigue syndrome:

Science is fully retracting the Report “Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome”.

He writes that the decision was reached because multiple laboratories have failed to reliably detect XMRV or related viruses in CFS patients. He also cites evidence of ‘poor quality control in a number of specific experiments in the report’, and that Figure 1, table S1, and figure S2 have been retracted by the authors. Finally, he notes the omission of information from the legend of figure 2C, specifically that the authors failed to indicate that the peripheral blood mononuclear cells had been treated with azacytidine, phytohemagglutinin, and IL-2. He concludes:

Given all of these issues, Science has lost confidence in the Report and the validity of its conclusions. We note that the majority of the authors have agreed in principle to retract the Report but they have been unable to agree on the wording of their statement. It is Science’s opinion that a retraction signed by all the authors is unlikely to be forthcoming. We are therefore editorially retracting the Report. We regret the time and resources that the scientific community has devoted to unsuccessful attempts to replicate these results.

Results of the Blood XMRV Scientific Research Working Group

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The Blood XMRV Scientific Research Working Group was formed to design and carry out a study to determine whether xenotropic murine leukemia virus-related virus (XMRV) posed a threat to blood safety. Phase III results were published on Sept. 22, 2011 in Science. In an upcoming webinar study leaders Graham Simmons and Mike Busch will present their findings and discuss their consequences for blood safety and the understanding of this agent’s role in chronic fatigue syndrome (CFS).

Please register to attend the webinar on 14 October 2011 at 4:00 PM EDT.

Update: Materials from the webinar, including the slides, a video of the presentation, and answers to 10 common criticisms of the study have been posted at Research1st.

Admit when you are wrong

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One of the lessons learned from XMRV is that it’s important for scientists to admit when they are wrong. That is why I took down the image originally posted with TWiV #150.

I had intended for the image to be a counterpoint to T-shirts worn by CFS patients proclaiming them to be ‘XMRV Positive’. I felt it was equally important to advertise the message that XMRV is a contaminant. It was not meant to be disparaging or humorous. However a number of individuals felt otherwise, and told me so in rather harsh terms. Then I received the following email:

I am writing with a concern about an image shown on your website/video blog TWiV.

I have been religiously following you since the first news of the findings in Lombardi et al. I regularly turn to your blog for real scientific information and not the hearsay and pseudo scientific nonsense that permeates the internet.

As a long term patient whose health is deteriorating, I find myself often discouraged by the levels that the conversation regarding CFS drops to. It seems hard to difficult at times scientists willing to work hard without prejudice towards a cure for this terrible disease.

The image in question serves only to widen the divide between patients and researchers. While people suffer, the scientific community has a chuckle at our expense.

I would ask that they image be removed and replaced with one of unity. While XMRV did not pan out, patients are still in need. We need to know the scientific community is doing all they can to save us.

None of the earlier comments that I received about the image were logical and composed; they brimmed with vile. This respectful and reasonable request convinced me that the image was not helpful, so I removed it.