TWiV Special: A paradigm for pathogen de-discovery

On this special episode of the science show This Week in Virology, Vincent and Ian review a multicenter blinded analysis which finds no association between chronic fatigue syndrome/myalgic encephalomyelitis and XMRV or polytropic murine leukemia virus.

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A viral mashup in snakes

snake inclusion body diseaseIf you know anything about snakes you might be familiar with snake inclusion body disease, or IBD. This transmissible and fatal disease affects snakes of a variety of species but has been best studied in boas. The name comes from the presence of large masses (inclusions) in the cytoplasm of cells from infected snakes. IBD might be caused by a novel arenavirus.

To identify an etiologic agent of IBD, RNA was extracted from multiple organs of snakes with the disease, and subjected to deep sequencing. This analysis revealed the presence of two distinct arenaviruses. One virus, called CASV (California Academy of Sciences virus) was found in diseased annulated tree boas, and the second, GGV (Golden Gate virus) was detected in boa constrictors. These sequences were found in 6 of 8 IBD snakes but not in 18 disease-free controls.

The finding of arenaviruses in snakes is interesting because these viruses are thought to infect only mammals. Rodents are believed to be the natural host of arenaviruses, which are classified as Old World or New World depending on where they are isolated. In rodents, arenavirus infection is typically asymptomatic. When arenaviruses infect humans, severe disease can result, such as hemorrhagic fever caused by Lassa virus. How CASV and GGV are transmitted to snakes is not known. One possibility is that they are introduced into snakes when they consume mice. The viruses might be transmitted among snakes by contact or via vectors such as blood-sucking mites. The genome sequences of CASV an GGV are very different from those of rodent arenaviruses. If similar viruses circulate in rodents, they have not yet been detected; alternatively, CASV- and GGV-like viruses might have diverged from Old- and New World arenaviruses after many years of transmission among snakes.

Another surprise emerged from analysis of the CASV and GGV viral proteins. Arenaviral genomes encode four main proteins: an RNA polymerase, L; a nucleoprotein, NP; a transmembrane glycoprotein, GPC, and a zinc-binding protein, Z. The amino acid sequences of CASV and GGV L, and NP, but not Z and GPC, resemble those of known arenaviruses. The CASV and GGV glycoproteins are instead related to glycoproteins of filoviruses and retroviruses. This observation suggests that recombination took place between the genomes and arenaviruses and filoviruses or retroviruses, likely a very long time ago.

Whether these novel arenaviruses actually cause snake IBD is not proven by this work. This question is underscored by the observation that no arenaviruses were detected in two of the 8 IBD positive snakes in this study. In addition, two of the virus-positive snakes that were diagnosed with IBD did not have symptoms of the disease. It is possible that the arenaviruses are present but do not cause symptoms. As the authors write,

….sequencing can only ever identify candidate etiologic agents, and demonstration of causality requires significant additional experimental effort.

This additional work would include the demonstration that infectious virus can be consistently recovered from diseased snakes, and that the disease can be induced by inoculation of snakes with the virus. As a first step towards answering these questions, kidney and liver extracts were added to cultured boa constrictor kidney cells. By 5 days post-infection, viral RNA could be detected in the cell supernatant, but it is not known if the viruses produced are infectious.

This work shows convincingly that the host range of arenaviruses is much broader than we thought: they do not just infect mammals. The zoonotic pool continues to grow, and there are now more potential sources of new human arenaviruses. The work also emphasizes that our knowledge of all the viruses on the planet remains miniscule.

Mark D. Stenglein, Chris Sanders, Amy L. Kistler, J. Graham Ruby, Jessica Y. Franco, Drury R. Reavill, Freeland Dunker, and Joseph L. DeRisi. 2012. Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease. mBio 3:e00180-12.

TWiV 194: Five postdocs in North America

On episode #194 of the science show This Week in Virology, Vincent returns to Madison, Wisconsin and meets with postdocs to discuss their science and their careers.

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Cleaning up after XMRV

XMRVThe retrovirus XMRV does not cause prostate cancer or chronic fatigue syndrome – that hypothesis was disproved by the finding that the virus was produced in the laboratory in the 1990s by passage of a prostate tumor in nude mice. A trio of new papers on the virus attempt to address questions about the serological detection of XMRV in prostate cancer, and further emphasize that XMRV is not a human pathogen.

Absence of XMRV and Closely Related Viruses in Primary Prostate Cancer Tissues Used to Derive the XMRV-Infected Cell Line 22Rv1. The human cell line 22Rv1, which was established from a human prostate tumor (CWR22), produces infectious XMRV. It was previously shown that DNA from various passages of the prostate tumor in nude mice (called xenografts), did not contain XMRV, but cells from the mice do contain two related proviruses called PreXMRV-1 and PreXMRV-2 which recombined to form XMRV between 1993-1996. In a new study samples of the original prostate tumor CWR22 were examined for the presence of XMRV or related viruses. PCR assays targeting the viral gag, pol, and env sequences failed to provide evidence of XMRV in CWR22 tissue. These assays could detect endogenous murine leukemia virus DNA in mouse DNA, indicating that the CWR22 tumor contained neither XMRV nor related viruses. In addition, no XMRV sequences were detected when sections from the CWR22 tumor were examined by in situ hybridization. The same assay previously detected XMRV sequences in stromal cells of prostate tumors. The authors conclude that “Our findings conclusively show an absence of XMRV or related viruses in prostate of patient CWR22, thereby strongly supporting a mouse origin of XMRV.”

An important question not addressed by this study is why XMRV was originally detected in multiple prostate tumors obtained from patients at the Cleveland Clinic. The authors seem to be working on this problem, as they state that “…the sequence of XMRV present in 22Rv1 cells is virtually identical with XMRV cloned using human prostate samples, thus suggesting laboratory contamination with XMRV nucleic acid from 22Rv1 cells as the source. Further experiments designed to confirm or refute this hypothesis are currently underway.”

No biological evidence of XMRV in blood or prostatic fluid from prostate cancer patients. Samples from individuals with prostate cancer were tested for the presence of infectious XMRV and for antibodies against the virus. Neither infectious virus nor antibodies were detected in blood plasma (n = 29) or prostate secretions (n = 5). Among these were five specimens that had previously tested positive for XMRV DNA, including two from the original study. The authors conclude that the results “support the conclusion from other studies that XMRV has not entered the human population”.

Susceptibility of human lymphoid tissue cultured ex vivo to Xenotropic murine leukemia virus-related virus (XMRV) infection. Although XMRV is not known to cause human disease, whether it has to potential to do so is unknown. The virus can infect a variety of cultured human cells including peripheral blood mononuclear cells and neuronal cells. In this study the authors placed human tonsillar tissue in culture and infected it with XMRV. Proviral (integrated) DNA could be detected in the cells several weeks after infection and virus particles were released into the medium. However these released viruses could not infect fresh tonsillar tissue, possibly due to modification by innate antiviral restriction factors such as APOBEC, which is known to inhibit XMRV infectivity.

Based on their findings the authors conclude that “laboratories working with XMRV producing cell lines should be aware of the potential biohazard risk of working with this replication-competent retrovirus”.

It is clear that XMRV does not cause chronic fatigue syndrome; the original findings of Lombardi and colleagues linking the virus to this disease have been retracted by the journal. However there are still two papers in the literature that report the presence of XMRV in prostate – the original XMRV discovery paper and one from Ila Singh’s laboratory. In both papers XMRV detection in tissues was accomplished by using serological procedures. Based on the papers summarized here, the assays did not detect XMRV – but a satisfactory explanation for the positive signals has not yet been provided.

TWiV 174: Dog runs and mooing miRs

On episode #174 of the podcast This Week in Virology, Vincent, Alan, and Rich consider whether pet dogs might transmit human noroviruses, and an RNA virus microRNA that might be involved in oncogenesis.

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TWiV 164: Six steps forward, four steps back

xmrvHosts: Vincent RacanielloRich 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)

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Rich – Fundamentals of Molecular Virology by Nicholas H. Acheson
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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.

Science retracts paper on detection of XMRV in CFS patients

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.

TWiV 158: Wolverines go viral

poliovirus + CD155Hosts: Vincent RacanielloRich Condit, Alice Telesnitsky, and Kathy Spindler

Vincent and Rich visit the Microbiology and Immunology Department at the University of Michigan Medical School, and speak with Alice and Kathy about their work on HIV genome dimerization, and packaging and pathogenesis of mouse adenovirus.

Model of poliovirus bound to CD155 made by Stefan Taube.

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Alice – Banvard’s Folly by Paul S. Collins
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TWiV 157: Better innate than never

dendritic cellHosts: Vincent RacanielloRich Condit, Alan Dove, Dickson Despommier, Jeremy Luban, and Gabriel Victora

A large TWiV panel remembers Ralph Steinman, and considers a new innate sensor of retroviral capsids.

Photograph of a dendritic cell (green) interacting with T cells (cyan) near a blood vessel by Gabriel Victora.

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Dickson – Eats, Shoots & Leaves by Lynne Truss
Rich – Battlestar Galactica
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DavidCarl Sagan’s Pale Blue Dot (YouTube)

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