TWiV 320: Retroviruses and cranberries

On episode #320 of the science show This Week in Virology, Vincent speaks with John Coffin about his career studying retroviruses, including working with Howard Temin, endogenous retroviruses, XMRV, chronic fatigue syndrome and prostate cancer, HIV/AIDS, and his interest in growing cranberries.

You can find TWiV #320 at

TWiV 213: Not bad for a hobby

On the final episode of the year of the science show This Week in Virology, the TWiV team reviews twelve cool virology stories from 2012.

You can find TWiV #213 at

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 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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Click the arrow above to play, or right-click to download TWiV 164 (60 MB .mp3, 99 minutes).

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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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GarrenTrillion-frame-per-second video
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Brain Picking’s 11 best science books of 2011

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TWiV 123: Contaminated prostates, absolute truth, and bleached worms

42Hosts: Vincent Racaniello, Alan Dove, and Rich Condit

On episode #123 of the podcast This Week in Virology, Vincent, Alan, and Rich talk about XMRV integration sites in prostate tumor DNA, the decline effect and scientific method, and the first virus of Caenorhabditis nematodes.

Click the arrow above to play, or right-click to download TWiV #123 (67 MB .mp3, 93 minutes).

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Rich – Einstein: His Life and Universe by Walter Isaacson (Aspen Institute)
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Authenticity of XMRV integration sites

retroviral integrationIntegration of retroviral DNA into the cellular genome is essential for the production of new infectious particles. A strong argument that the novel human retrovirus XMRV is not a laboratory contaminant is the finding that viral DNA is integrated in chromosomal DNA of prostate tumors. Nucleotide sequence analyses of 14 integration sites in prostate tumor DNAs from 9 different patients previously revealed the expected viral sequences linked to human DNA. But two of these integration sites are identical to those found in a prostate tumor cell line infected with XMRV.

A search of the nucleotide sequence database with the previously identified XMRV integration site sequences revealed that 2 of the 14 sequences (from 2 patients) were identical to two XMRV integration sites in DU145 cells. This cell line was established in 1978 from the brain metastasis of a human prostate tumor. In early 2010 2007 DU145 cells were infected with XMRV, and sequences of two integration sites were determined (the database entries can be found here and here).

Identical retroviral integration sites have never been reported in independently infected cells. Furthermore, XMRV infection of DU145 cells was done in the same laboratory in which the XMRV integration sites were identified in prostate tumor DNA. The conclusion is that two of the 14 XMRV integration sites in prostate tumor DNA are likely to be the result of contamination. These prostate tumor DNA samples were probably contaminated with DNA from XMRV-infected DU145 cells.

These observations do not directly impugn the veracity of the other 12 XMRV integration sites identified in prostate tumor DNA. However, when DNA contamination occurs it is often ubiquitous. Hence the authors write:

Whilst it is conceivable that the other 12 integration sites apparently derived from prostatic tumor tissues are genuine patient-derived sequences, we suspect that some or all of them may also be the result of contamination with DNA from experimentally infected DU145 cells.

This possibility can and must be addressed experimentally.

Update: While writing this post I received an abstract from the 2011 Conference on Retroviruses and Other Opportunistic Infections (CROI) entitled “XMRV probably originated through recombination between two endogenous murine retroviruses during passage of a human prostate tumor in nude mice”. As usual I will await publication of this story in a peer-reviewed journal before discussing it further.

Garson JA, Kellam P, & Towers GJ (2011). Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection. Retrovirology, 8 (1) PMID: 21352548

Stone, K., Mickey, D., Wunderli, H., Mickey, G., & Paulson, D. (1978). Isolation of a human prostate carcinoma cell line (DU 145) International Journal of Cancer, 21 (3), 274-281 DOI: 10.1002/ijc.2910210305

Dong B, Kim S, Hong S, Das Gupta J, Malathi K, Klein EA, Ganem D, Derisi JL, Chow SA, & Silverman RH (2007). An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors. Proceedings of the National Academy of Sciences of the United States of America, 104 (5), 1655-60 PMID: 17234809

Retroviral integration and the XMRV provirus

XMRVA strong argument that the novel human retrovirus XMRV is not a laboratory contaminant is the finding that viral DNA is integrated in chromosomal DNA of prostate tumors. Why does this result constitute such strong proof of viral infection?

Establishment of an integrated copy of the viral genome – the provirus – is a critical step in the life cycle of retroviruses. Proviral DNA is transcribed by cellular RNA polymerase II to produce the viral RNA genome and the mRNAs required to complete the replication cycle. Without proviral DNA, retroviral replication cannot proceed.

To produce proviral DNA, the retroviral RNA genome is converted to a double-stranded DNA by the viral enzyme reverse transcriptase. This step occurs in the cytoplasm. Specific and efficient insertion of the viral DNA into the host cell DNA is catalyzed by a viral enzyme called integrase. This enzyme recognizes and nicks the two ends of viral DNA, and the new 3′-ends are then joined covalently to the host DNA at staggered nicks made by integrase.

The image below shows some of the characteristic features of retroviral integration. A the top is the unintegrated linear DNA of avian sarcoma/leukosis virus produced by reverse transcription. Upon completion of integration, two base pairs (AA•TT) are lost from both termini, and a 6-bp target site in host DNA (pink) is duplicated on either side of the proviral DNA. This target site varies in length from 4 to 6 bp among different retroviruses. The proviral DNA (middle) ends with the conserved 5′-T G…C A-3′ sequence. The provirus serves as a template for the production of the viral RNA genome (bottom).

To identify XMRV proviral DNA, genomic DNA was isolated from prostate tumors, and DNA was amplified using a primer that annealed in the viral env gene, near the right-hand LTR. Nucleotide sequence analyses of amplified DNAs from 14 9 different patients showed the expected viral CA sequence followed by human DNA. However, the other cardinal sign of retroviral integration – duplication of host DNA sequences flanking the integration site – could not be confirmed, because only the right-hand integration site was studied.

The isolation of the entire proviral DNA, including both flanking integration sites, from patients with prostate cancer or chronic fatigue syndrome would be additional evidence that XMRV is a virus that infects humans.

Kim, S., Kim, N., Dong, B., Boren, D., Lee, S., Das Gupta, J., Gaughan, C., Klein, E., Lee, C., Silverman, R., & Chow, S. (2008). Integration Site Preference of Xenotropic Murine Leukemia Virus-Related Virus, a New Human Retrovirus Associated with Prostate Cancer Journal of Virology, 82 (20), 9964-9977 DOI: 10.1128/JVI.01299-08

TWiV 113: Alan Rein on XMRV

Hosts: Vincent RacanielloAlan DoveRich Condit, and Alan Rein

rich conditOn episode #113 of the podcast This Week in Virology, Vincent, Alan, and Rich discuss the retrovirus XMRV with retrovirologist Alan Rein of the National Cancer Institute.

Click the arrow above to play, or right-click to download TWiV #113 (55 MB .mp3, 76 minutes).

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XMRV and CFS – It’s not the end

Yesterday the Chicago Tribune published my reaction to the four papers on the retrovirus XMRV published this week in the journal Retrovirology. I was quoted as saying “These four papers are probably the beginning of the end of XMRV and CFS”. I wish to retract this statement and explain my reasons for doing so.

Early Monday a reporter for the Chicago Tribune, Trine Tsouderos, sent an email asking for my thoughts on four XMRV papers that had just been released (paper one, two, three, four). I read all four papers and decided that they raised serious concerns about the role of XMRV in human disease. Specifically, the four papers demonstrated different ways that assays for XMRV could be subject to contamination with murine viral sequences. I wrote an email to Ms. Tsouderos outlining my summary of the papers, and later that day her article was published. My statement was reproduced exactly from the email I had sent her, so I was not misquoted.

I then set out to write about the papers for my blog about viruses. I read the papers over again, and began checking XMRV sequences in Genbank. I also began an email correspondence with authors of three of the four papers, and spoke with my virology colleagues here at Columbia. As a consequence of this additional research I decided that my initial impression of the papers was incorrect, which is evident in my post entitled ‘Is XMRV a laboratory contaminant?‘. Almost immediately after publishing the piece readers began to ask why my comments to the Chicago Tribune had such a different tone. I concluded that a retraction and explanation were necessary.

Upon re-reading three of the four Retrovirology papers it became clear to me that they show that identification of XMRV can be fraught with contamination problems, but they do not imply that previously published studies are compromised by these findings. Clearly any new studies done on XMRV should keep in mind the potential for contamination from PCR kits and murine nucleic acids.

I was initially more troubled by the fourth paper by Hue and colleagues. There are four major findings in this paper (gag PCR primers are not specific for XMRV; the virus is present in 5 human tumor cell lines; two XMRV isolates are nearly identical to a virus from the human prostate cell line and also contain an insertion from the murine retrovirus MoMLV; and there is more nucleotide diversity in viral sequences from 22Rv1 cells than in all the patient XMRV sequences). The fact that two XMRV isolates seem to be laboratory contaminants – judged by the presence of MoMLV sequences – was initially unsettling until it became clear that other XMRV isolates do not have this insertion. That leaves the fourth finding – that XMRV from 22Rv1 cells appears ancestral to, and more diverse than, all the human XMRV sequences. I decided that this result was less troublesome than I had originally believed, in part because it is not clear that the differences among the 22Rv1 viruses did not arise during PCR amplification.

My conclusion is that these four papers point out how identification of XMRV from human specimens can be complicated by contamination, but they do not mean that previous studies were compromised. They serve as an important reminder that future experiments to identify XMRV need to be appropriately controlled to ensure that the results are not compromised by contamination.

In other words, these four papers are NOT the beginning of the end of XMRV and CFS. Rather, research on the role of this virus in human disease must proceed, with large, case-controlled epidemiological studies, as suggested by others.

I would like to apologize to anyone who was offended, angered, or disappointed in any way by my statement to the Chicago Tribune. It is my goal to educate the public about virology, and clearly I did not do that very well.

There are at least two lessons that you can take away from this incident. First, that I make mistakes, and that I’m willing to admit it. Everyone does, including scientists. Second, if I had difficulties interpreting these papers, how would non-scientists fare?

Is XMRV a laboratory contaminant?

XMRVSince 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.

Update #1: 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.

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