Results of the Blood XMRV Scientific Research Working Group

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

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.

Trust science, not scientists

XMRVWhether or not the retrovirus XMRV is a human pathogen has been debated since the virus was first described in 2006. The answer is now clear: the results of Blood XMRV Scientific Research Group, along with a partial retraction of the 2009 Science paper describing identification of the retrovirus in patients with chronic fatigue syndrome (CFS) show that detection of XMRV in patient samples is a result of contamination.

The Blood XMRV group obtained new blood samples from 15 individuals previously shown to be positive for XMRV (Lombardi et al., 2009) or MLV (Lo et al., 2010) ; 14 of these were from CFS patients. Fifteen blood samples were also obtained from healthy donors. The samples were coded and sent to 9 laboratories for analysis. These laboratories (Abbott Molecular, Abbott Diagnostics, CDC, FDA/Lo, FDA/Hewlett, Gen-Probe, NCI/DRP, and WPI) conducted validated assays for viral nucleic acid, viral replication, or viral antibodies. Positive control samples were also included that were ‘spiked’ with XMRV, in the form of cell culture fluids from the cell line 22Rv1. Each laboratory was at liberty to choose which assays to carry out.

Two laboratories reported evidence of XMRV in the coded samples.  Only WPI identified positive specimens by PCR: two from negative controls, and one from a CFS patient. The FDA/Lo laboratory did not detect any positives by PCR, using the same nested assay that they had previously reported in their published study. The samples tested included 5 specimens that were positive in the Lo et al. study.

Lombardi and colleagues have previously concluded that viral culture is the most sensitive method for detecting XMRV; however the FDA/Hewlett laboratory failed to culture virus from CFS samples. This laboratory did culture virus from positive control specimens, demonstrating the sensitivity of their methods. The FDA/Ruscetti laboratory recovered virus from 3/15 CFS samples but also from 6/15 negative control specimens. WPI did not carry out viral culture assays due to contamination of their cell lines with mycoplasma.

Four laboratories tested the samples for the presence of antibodies that react with XMRV proteins. Only WPI and NCI/Ruscetti detected reactive antibodies, both in CFS specimens and negative controls. There was no statistically significant difference in the rates of positivity between the positive and negative controls, nor in the identity of the positive samples between the two laboratories.

These results demonstrate that XMRV or antibodies to the virus are not present in clinical specimens. Detection of XMRV nucleic acid by WPI is likely a consequence of contamination. The positive serology reported by WPI and NCI/Ruscetti laboratories remained unexplained, but are most likely the result of the presence of cross-reactive epitopes. The authors of the study conclude that ‘routine blood screening for XMRV/P-MLV is not warranted at this time’.

One of the authors on Lombardi et al., Robert Silverman, decided to reexamine some of the DNA preparations from CFS patients that were originally used to detect XMRV DNA by PCR. He found that all the positive specimens from CFS patients were contaminated with XMRV plasmid DNA. Therefore the authors of the original study have retracted Figure 1 (single-round PCR detection of XMRV in CFS PBMC DNA); table S1, XMRV sequences, and figure S2, phylogenetic analysis of XMRV sequences.

A puzzling aspect of Silverman’s results is that XMRV plasmid DNA was detected only in samples from CFS patients, not healthy controls. This pattern would not be expected if the specimens were properly blinded, that is, coded so that the investigators did not know which were controls and which were from CFS patients. The authors offer no explanation of these findings.

The paper reporting contamination of samples with XMRV is entitled ‘Partial Retraction‘. It’s not clear to me why the entire paper has not been retracted. After removing the PCR and nucleic acid sequencing results, there is no evidence indicating the presence of XMRV in the patient samples. The remaining experiments show detection of a retrovirus by cell culture experiments, and the presence of viral proteins or antibodies to the virus in clinical specimens. None of these findings prove that what is being studied is XMRV. The title of the original paper ‘Detection of an infectious retrovirus’, XMRV, in blood cells of patients with chronic fatigue syndrome‘, is unsupported.

In an accompanying article on the XMRV story entitled ‘False Positive‘, Judy Mikovits of WPI notes that “Anyone who says this is a lab contaminant has drawn the wrong conclusion and has done a disservice to the public”. She goes on to imply that a gammaretrovirus is likely involved in CFS. On the contrary, pursuing the CFS-gammaretrovirus hypothesis is a disservice to those with CFS, and detracts from efforts to solve the disease. There are no data to support such an association, and to suggest that a lab contaminant, XMRV, has pointed the way to a bona fide etiologic agent seems implausible.

XMRV does not cause CFS. The virus arose in mice between 1993-96, and its detection in patient samples is clearly a result of contamination. Reaching these conclusions has required a long and often contentious journey that has highlighted the best and worst aspects of scientific research. There are many lessons to be learned from XMRV, but an important one is that science progresses not from the work of a single investigator, but from the collective efforts of many laboratories. XMRV reminds us to trust science, not scientists.

TWiV 150: Contaminated

pXMRVHosts: Vincent RacanielloRich Condit, and Dickson Despommier

Vincent, Dickson, and Rich meant to do an all-email episode, but first they review results of the Blood XMRV Scientific Research Working Group, and partial retraction of the paper associating XMRV with chronic fatigue syndrome.

With this episode TWiV is three years old.

Click the arrow above to play, or right-click to download TWiV 150 (56 MB .mp3, 93 minutes).

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Links for this episode:

Weekly Science Picks

Dickson – The Tree of Life
Vincent –
When do you fact-check article content with sources? (take as directed)
Rich –
io9

Listener Pick of the Week

LuisNIH videocasting and podcasting

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Live chat on XMRV and chronic fatigue syndrome

Science Live, the weekly chat at Science magazine, will tackle XMRV and chronic fatigue syndrome on Thursday, 22 September. From the Science Live webpage:

Two years ago, Science published a paper suggesting that a mouse retrovirus called XMRV might be involved in chronic fatigue syndrome (CFS), a debilitating disease with no known cause. The study raised worries that XMRV might be spreading via blood donations. Since then, many other studies have failed to find XMRV in CFS patients, and some have suggested that the 2009 paper was the result of lab contamination.

Michael Busch, a transfusion medicine scientist involved in XMRV research, and retrovirologist Jay Levy will be in the chat to answer questions.

Murine gammaretroviruses in prostate cancer cell lines

nude mouseThe retrovirus XMRV arose during passage of a human prostate tumor in nude mice. The genomes of these mice contain two different proviral DNAs related to XMRV, pre-XMRV-1 and pre-XMRV-2, that recombined to produce XMRV that has been isolated from humans. Two other prostate cancer cell lines also contain mouse gammaretroviruses that are not XMRV. These viruses may have entered the prostate tumor cells during xenograft passage in immunocompromised mice.

The discovery of infectious XMRV in the prostate tumor cell line 22Rv1 prompted the examination of other prostate tumor cell lines for the presence of murine gammaretroviruses. Antisera against Moloney murine leukemia virus were used to screen 72 cell lines by immunohistochemistry for the presence of murine gammaretroviruses. Three human prostate tumor cell lines (22Rv1, LAPC4, and VCaP) and one lung carcinoma cell line (EKVX) reacted with the antisera.

Polymerase chain reaction and nucleotide sequencing analysis revealed that these viruses are not XMRV. The virus in the EKVX cell line is a xenotropic MLV similar to a virus previously isolated from a human B-lymphoblastoid cell line. The virus from the LAPC4 and VCaP cell lines is the murine xenotropic retrovirus Bxv-1. A different sample of VCaP cells obtained from the ATCC were also positive for Bxv-1, as were LAPC4 cells obtained from a different laboratory. Replication-competent viruses were detected in all three cell lines.

How did these human prostate cancer cell lines become contaminated with murine gammaretroviruses? The authors believe this is a consequence of passage of the tumors through immunocompromised mice. In support of this hypothesis, the retrovirus Bxv-1 is present in nude mice, and passage of tumors in these mice can lead to infection with xenotropic MLVs. In contrast to the origin of XMRV, recombination was not needed to produce these viruses.

These findings led the authors to examine two other prostate cancer cell lines, present in their laboratory, DU145 and LNCaP, for the presence of gammaretroviruses. They found these cell lines to be contaminated with XMRV, likely obtained from 22RV1 cells in use in the laboratory. Fresh aliquots of the DU145 and LNCaP cells obtained from other sources were not contaminated. The authors conclude that if

CWR22Rv1 cells are routinely cultured in a typical biomedical research laboratory setting (e.g. using standard Class II biosafety cabinets and procedures for cell culture in which two different cell lines are never present under the hood at the same time), that XMRV can infect and contaminate other cell lines.

Karen Sandell Sfanos, Amanda L. Aloia, Jessica L. Hicks, David M. Esopi, Jared P. Steranka, Wei Shao, Silvia Sanchez-Martinez, Srinivasan Yegnasubramanian, Kathleen H. Burns, Alan Rein, & Angelo M. De Marzo (2011). Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines PLoS One : 10.1371/journal.pone.0020874

TWiV 136: Exit XMRV

nude mouseHosts: Vincent Racaniello, Alan Dove, Rich Condit, and Stephen Goff

Retrovirologist Stephen Goff joins Vincent, Rich, and Alan for a discussion of recent papers on the retrovirus XMRV and its association with chronic fatigue syndrome and prostate cancer.

Click the arrow above to play, or right-click to download TWiV #136 (61 MB .mp3, 84 minutes).

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Alan – The Demon-Haunted World by Carl Sagan
Rich
– Trends in annual rates of death (pdf)
Stephen – Unexpected inheritance (PLoS Pathogens)
Vincent – Beyond the human eye

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Raihan  – Bruce Aylward: How we’ll stop polio for good (YouTube)

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XMRV is a recombinant virus from mice

recombinant xmrvThe novel human retrovirus XMRV has been associated with prostate cancer and chronic fatigue syndrome. The nucleotide sequence of XMRV isolated from humans indicates that the virus is nearly identical with XMRV produced from a human prostate tumor cell line called 22Rv1. This cell line was derived by passage of human prostate tumor tissue in nude mice. Sequence analyses reveal that the genomes of these mouse strains contain two different proviral DNAs related to XMRV. These viral genomes recombined to produce XMRV that has been isolated from humans.

XMRV was originally isolated from a human prostate cancer in 2006, and subsequently associated with ME/CFS. The human cell line 22Rv1, which was established from a human prostate tumor (CWR22), produces infectious XMRV. An important question is whether XMRV was present in the original prostate tumor, or was obtained by passage through nude mice. To answer this question, DNA from various passages of the prostate tumor in nude mice (called xenografts), and the mouse strains used to passage the tumor, were analyzed for the presence of XMRV proviral DNA.

Early-passage xenografts did not contain XMRV, but mouse cells found in them did contain two related proviruses called PreXMRV-1 and PreXMRV-2. The 3’-3211 nucleotides of PreXMRV-1, and both LTRs, are identical to XMRV save for two nucleotide differences. The genomic 5’-half of XMRV and PreXMRV-1 differs by 9-10%. PreXMRV-1 is defective for replication due to mutations in genes encoding the gag and pol proteins. PreXMRV-2 does not contain obvious mutations that would prevent the production of infectious viruses. The gag-pro-pol and a part of the env region of this viral genome is identical to that of XMRV save for two base differences; the LTRs and the remainder of the genome differ by 6-12% from XMRV.

Comparison of the sequences of PreXMRV-1 and PreXMRV-2 indicates that recombination between the two viral genomes led to the formation of XMRV. When the sequences of PreXMRV-1 and −2 are used to construct the recombinant XMRV, the resulting virus differs by only 4 nucleotides from the consensus XMRV sequence derived from all human isolates reported to date.

The nude mice used for passage of the original prostate tumor were likely the NU/NU and Hsd strains. Neither mouse strain contains XMRV proviral DNA, but both contain PreXMRV-1 and PreXMRV-2 proviral DNA.

These data demonstrate that XMRV was not present in the original CWR22 prostate tumor, but arose by recombination of PreXMRV-1 and PreXMRV-2 between 1993-1996. When the original prostate tumor was implanted into nude mice, some of the mice harbored both pre-XMRV-1 and −2 endogenous proviruses, which recombined to form XMRV. The authors believe that XMRV originating from the CRWR22 xenografts, the22Rv1 cell line, or other related cell lines has contaminated all human samples positive for the virus. In addition, they suggest that PCR assays for XMRV may actually detect PreXMRV-1 and −2 or other endogenous viral DNA from contaminating mouse DNA.

Another possibility to explain the origin of XMRV is that it arose in mice and can infect humans. If this is true, then XMRV would have to be present in the nude mice used to passage the CWR22 human prostate tumor. No evidence for an XMRV provirus was found in 12 different nude mouse strains, including two used to passage the CWR22 tumor. Furthermore, a screen of 89 inbred and wild mice failed to reveal the presence of proviral XMRV DNA. Hence the authors conclude:

…that XMRV arose from a recombination event between two endogenous MLVs that took place around 1993-1996 in a nude mouse carrying the CWR22 PC xenograft, and that all of the XMRV isolates reported to date are descended from this one event.

It is possible that XMRV produced during passage of CWR22 in nude mice subsequently infected humans. Because XMRV arose between 1993-1996, this scenario could not explain cases of prostate cancer and chronic fatigue syndrome that arose prior to that date.

How can these findings be reconciled with the published evidence that sera of ME/CFS patients from the 1980s contain antibodies to XMRV? Those antibodies were not shown to be directed specifically against XMRV, and therefore cannot be used to prove that XMRV circulated in humans prior to 1993-96. Furthermore, in the absence of clear isolation of an infectious virus, antibody tests alone have proven highly unreliable for identification of new viruses.

Where do these findings leave the hypothesis that XMRV is the etiologic agent of prostate cancer and ME/CFS? All published sequences of human XMRV isolates are clearly derived by recombination of PreXMRV-1 and −2. The finding of human XMRV isolates that are not derived from PreXMRV-1 and −2 would leave a role for XMRV in human disease. As of this writing, no such XMRV isolates have been reported in the scientific literature.

Update: A second paper has also been published in Science Express today entitled “No evidence of murine-like gammaretroviruses in CFS patients previously identified as XMRV-infected”. Editors of the journal Science have asked the authors to retract their 2009 paper linking XMRV infection with chronic fatigue syndrome. The authors have refused.

T. Paprotka, K. A. Delviks-Frankenberry, O. Cingoz, A. Martinez, H.-J. Kung, C.G. Tepper, W-S Hu, M. J. Fivash, J.M. Coffin, & V.K. Pathak (2011). Recombinant origin of the retrovirus XMRV. Science Express

Ian Lipkin on XMRV

XMRVLate last year virologist Ian Lipkin was asked by National Institute of Allergy and Infectious Diseases head Anthony Fauci to coordinate a multi-center study of CFS patients. Newly drawn blood samples from 100 CFS patients and 100 healthy controls from around the US will be blinded and sent to three groups – FDA, CDC and the Whittemore Peterson Institute – and assayed for the presence of XMRV. After the recent publication by Ila Singh on XMRV in CFS patients, Dr. Lipkin sent me the following note:

Dear Vince-

We have a plethora of explanations for how CFS/XMRV/MLV studies could go awry. However, we don’t have evidence that they have. Absent an appropriately powered study representing blinded analyses by Mikovitz and Lo/Alter of samples from well characterized subjects using their reagents, protocols and people, all we have is more confusion.

I remain agnostic. We won’t have answers until the end of 2011.

The NIH will post something on our study today.

Ian

Ila Singh finds no XMRV in patients with chronic fatigue syndrome

XMRVSince the first association of the retrovirus XMRV with chronic fatigue syndrome in 2009 in the US, subsequent studies have failed to detect evidence of infection in patients from the US, Europe, and China. These studies were potentially compromised by a number of factors, such as differences in patient characterization, geographic locations, clinical samples used, and methods used to detect the virus. These and other potentially confounding conditions have been addressed in the most comprehensive study to date on the association of XMRV with CFS.

In the introduction to their paper, published in the Journal of Virology, the authors note other problems with many of the studies of XMRV in CFS patients:

  • Too small control populations
  • Patient and control samples collected at different times
  • Investigators generally not blinded to sample identity
  • PCR assays that rely on conservation of viral sequence mainly used
  • Limits of detection, reproducibility, and precision of assays unknown
  • Controls for each step that would identify analysis not done
  • Insufficient numbers of negative controls included
  • No study included positive samples from the original 2009 patient cohort of Lombardi et al.

To address these issues, the authors collected blood from 105 CFS patients and 200 healthy volunteers in the Salt Lake City area. One hundred of the patients fulfilled both the CDC-Fukuda and the Canadian consensus criteria for diagnosis of ME/CFS. The patients were selected from a clinic that specializes in the diagnosis and management of CFS and fibromyalgia.

New blood samples were also collected (by a third party) from 14 patients from the original study by Lombardi et al. The samples were blinded for subsequent study. Detection of viral nucleic acids was done using four different PCR assays. Anti-XMRV antibodies in patient sera were detected by ELISA. Finally, virus growth from clinical specimens was attempted in cell culture. The authors used the multiple experimental approaches reported by Lombardi and colleagues.

Let’s go through the results of each assay separately.

PCR for viral nucleic acids. Four different quantitative PCR assays were developed that detect different regions of the viral genome. The assay for pol sequences has been used by several groups and is the most specific PCR assay for XMRV. Three other PCR assays were also used that target the LTR, gag and env regions of XMRV DNA. These assays could detect at least 5 viral copies of XMRV DNA. The precision and reproducibility of the PCR assays, as well as their specificity for XMRV, were also demonstrated. DNA prepared from white blood cells of 100 CFS patients and 200 controls were negative for XMRV. For every 96 PCR reactions, 12 water controls were included; these were always negative for XMRV DNA.

XMRV antibodies in human sera. To detect XMRV antibodies in human serum, a portion of the viral envelope protein, called SU, was expressed in cells and purified from the cell culture medium. The SU protein was attached to plastic supports, and human serum was added. Any anti-XMRV antibodies in human sera will attach to the SU protein and can subsequently be detected by a colorimetric assay (we have discussed this type of assay previously). This assay revealed no differences in the amount of bound human antibodies for sera from CFS patients or healthy controls. Some of the patient sera were also used in western blot analysis. Recombinant XMRV SU protein was fractionated by gel electrophoresis. The protein on the gel is then transferred to a membrane which is mixed with human serum. If there are anti-XMRV antibodies in the human serum, they will react with the SU protein on the membrane, and can be detected by a colorimetric assay. When rabbit anti-XMRV serum was used in this assay, the SU protein was readily detected. None of the human sera analyzed by this method were found to contain antibodies that detect SU protein.

Infectious XMRV in human plasma. It has been suggested that the most sensitive method for detecting XMRV in patients is to inoculate cultured cells with clinical material and look for evidence of XMRV replication. The XMRV-susceptible cell line LNCaP was therefore infected with 0.1 ml of plasma from 31 patients and 34 healthy volunteers; negative and positive controls were also included. Viral replication was measured by western blot analysis and quantitative PCR. No viral protein or DNA was detected in any culture after incubation for up to 6 weeks.

Analysis of previously XMRV-positive samples. Blood was drawn from twenty-five patients who had tested positive for XMRV as reported by Lombardi et al. These samples were all found to be negative for XMRV DNA and antibodies by the PCR and ELISA assays described above. In addition, no infectious XMRV could be cultured from these 25 samples.

Presence of mouse DNA. After not finding XMRV using qPCR, serological, and viral culture assays, the authors used the nested PCR assay described by Lo et al. Although positives were observed, they were not consistent between different assays. This led the authors to look for contamination in their PCR reagents. After examination of each component, they found that two different versions of Taq polymerase, the enzyme used in PCR assays, contained trace amounts of mouse DNA.

Given the care with which these numerous assays were developed and conducted, it is possible to conclude with great certainty that the patient samples examined in this study do not contain XMRV DNA or antibodies to the virus. It’s not clear why the 14 patients resampled from the original Lombardi et al. study were negative for XMRV in this new study. The authors suggest one possibility: presence of “trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies”. I believe that it is important to determine the source of XMRV in samples that have been previously tested positive for viral nucleic acid or antibodies. Without this information, questions about the involvement of XMRV in CFS will continue to linger in the minds of many non-scientists.

At the end of the manuscript the authors state their conclusion from this study:

Given the lack of evidence for XMRV or XMRV-like viruses in our cohort of CFS patients, as well as the lack of these viruses in a set of patients previously tested positive, we feel that that XMRV is not associated with CFS. We are forced to conclude that prescribing antiretroviral agents to CFS patients is insufficiently justified and potentially dangerous.

They also note that there is “still a wealth of prior data to encourage further research into the involvement of other infectious agents in CFS, and these efforts must continue.”

Clifford H. Shin, Lucinda Bateman, Robert Schlaberg, Ashley M. Bunker, Christopher J. Leonard, Ronald W. Hughen, Alan R. Light, Kathleen C. Light, & Ila R. Singh1* (2011). Absence of XMRV and other MLV-related viruses in patients with Chronic Fatigue Syndrome. Journal of Virology : 10.1128/JVI.00693-11