Monday, February 15, 2010

XMRV Discussion on Faculty of 1000

In a recent blog posting discussing the pros and cons of the two
currently published studies on XMRV and CFS, which itself is one of
the better reviews I've read, the author refers to a post in a
discussion on Faculty of 1000, which appears to be a kind of forum for
professionals to comment on research papers. There are several
comments made, including one dissenting opinion and an author's
response by Dr. Mikovits. There's not really any back and forth
discussion that I've seen; it's mainly straight commenting. The
comments made thus far are copied and pasted below.

Blog post-

Link to Faculty of 1000 Biology comments on 'Detection of an
infectious retrovirus, XMRV, in blood cells of patients with chronic
fatigue syndrome'-
Link to Faculty of 1000 Medicine comments on 'Detection of an
infectious retrovirus, XMRV, in blood cells of patients with chronic
fatigue syndrome'-

Faculty of 1000 Biology comments on 'Detection of an infectious
retrovirus, XMRV, in blood cells of patients with chronic fatigue

Ulf Pettersson
Uppsala University, Sweden
Genomics & Genetics

Chronic Fatigue Syndrome (CFS) is an enigmatic disease with an unknown
pathogenicity. In this paper, the authors have tested the hypothesis
that a human gamma retrovirus (XMRV) is a causative agent. The authors
analysed blood from 101 CFS patients and 218 controls. A dramatic
difference was found as 67% of the patients were positive for XMRV
whereas only 3.7% of the healthy controls were positive for the same

Moreover, the virus was infectious when tested in tissue culture. The
authors conclude with caution that XMRV could be a contributing factor
in the pathogenesis of CFS.
The reported results are undoubtedly
convincing. They should, however, be interpreted with caution as CFS
patients might have a malfunctioning immune system and the virus might
then be an innocent passenger. An interesting implication of the
finding is that XMRV has been suspected to be involved in prostate
cancer and it will be interesting to determine what connection there
could be between CFS and prostate cancer.

Competing interests: None declared
Evaluated 13 Nov 2009

Alan Landay
Rush University Medical Center, United States of America
Pharmacology & Drug Discovery

Lombardi and colleagues have isolated DNA from xenotropic murine
leukemia virus-related virus (XMRV) from the cells of patients with
chronic fatigue syndrome (CFS) and have found antibodies to this
retrovirus in their plasma.

CFS is a disease that has been studied for many years by the medical
community which is diagnosed most often by exclusion. It clearly
affects the immune system, with a response characterized by chronic
immune activation and a reduction in natural killer cell function. A
number of viruses (herpes and enterovirus) have been implicated as an
etiologic agent in CFS but none have been conclusively associated with
the disease. The authors of the current study demonstrate that a large
proportion of CFS patients are infected with XMRV (67%) and in vitro
both T and B cells can be infected with XMRV. The results of this
paper raise a number of interesting questions on the role of XMRV in
the pathogenesis of CFS and the diagnosis and treatment of CFS.

Competing interests: None declared
Evaluated 16 Nov 2009

Brigitte Huber
Tufts University School of Medicine, United States of America

This paper shows that a high frequency of chronic fatigue syndrome
(CFS) patients are infected with a xenotropic murine leukemia-like
virus (XMRV), compared to only a few normal controls. This is
particularly exciting because CFS is ill-defined and has been
approached with hesitation by scientists.

For the first time, we now have evidence that an infectious agent is
associated with this chronic disease. It remains to be shown whether
XMRV is directly responsible for the symptoms, or induces expression
of other cellular genes that lead to an inflammatory response.
Regardless, these new findings open the door for therapeutic
intervention of this dreaded disease.
This same virus has also been
associated with highly malignant prostate cancer {1}, but, again, the
significance of those results is not yet understood.

Competing interests: None declared
Evaluated 20 Oct 2009

Follow-up: IN RESPONSE TO DISSENTING OPINION: I agree with all the
points raised by Patrick Moore. Unless the published results can be
reproduced in an independent lab, we have to use caution with the
interpretation of the presented data.

Evaluated 18 Nov 2009

Adriano Boasso and
Gene Shearer
National Cancer Institute, National Institutes of Health, United
States of America

During the past several years, a debate has raged concerning whether
Chronic Fatigue Syndrome (CFS) is a virally induced condition, or even
a medically relevant phenomenon. This publication is exceptional
because it uniquely identifies, in CFS patients, a human
gammaretrovirus that is a xenotropic murine leukemia virus-related
virus (XMRV), which was recently discovered in a subset of prostate
cancer patients.

In this report, the DNA from XMRV was detected in the blood of 68/101
(67%) CFS patients, but only in 8/218 (3.7%) healthy controls. The env
and gag viral sequences of all positive patients were >99% similar to
those from tumor tissue of XMRV-positive prostate cancer patients. The
possibility that the XMRV genomes were murine leukemia virus (MLV)
contaminants was excluded by the finding that the CFS XMRV sequences
clustered with those from prostate cancer patient XMRV rather than
those from MLV. XMRV proteins were produced by activated T and B
lymphocytes from CFS patients, confirming that both of these cell
subsets were productively infected. Transmission of cell-associated
XMRV was observed in co-culture with activated T cells from CFS and
prostate cancer patients or T cell lines. Cell-free XMRV transmission
(from patients' activated T cell cultures) was demonstrated in primary
T cells from healthy controls. Finally, the plasma of 9/18 patients
compared to the plasma of 0/9 controls contained XMRV antibodies that
exhibited blocking activity, demonstrating that patients can elicit a
virus-specific immune response. The authors carefully emphasize that
their findings indicate an association between XMRV and CFS, and not
necessarily a cause-and-effect relationship.
Furthermore, this
discovery and the virologic and immunologic parameters analyzed here,
as well as the connection with prostate cancer, provide many
intriguing areas for future research. These include the molecular
retrovirology of XMRV and its susceptibility to antiretrovirals, the
mode(s) of XMRV transmission, the genetic susceptibility of
individuals to the virus, how infection by this virus can be
diagnosed, how XMRV is associated with cancer, the immunological
processes involved in XMRV infection, and the development of potential

Competing interests: None declared
Evaluated 21 Oct 2009

Follow-up: Nevertheless, it is important to consider that criticisms
have been made which raise skepticism on the results presented in the
study (see dissenting evaluation). The detection of XMRV in CFS
patients should be confirmed by other groups that have expertise in
XMRV-related virology and CFS, as well as in other cohorts, before
full conclusions can be made on the scientific and medical
implications of these findings.

Evaluated 18 Nov 2009

Katie Kelm and
George Breese
University of North Carolina at Chapel Hill, United States of America
Pharmacology & Drug Discovery

When one considers how little is known about chronic fatigue syndrome
(CFS), Lombardi and colleagues have made a major breakthrough in
understanding CFS with the discovery of a highly significant
association between CSF and the xenotropic murine leukemia
virus-related virus (XMRV).

CFS is a disease affecting roughly 17 million people worldwide and
involves multiple symptoms involving the nervous, endocrine and immune
systems {1}.
The cause of CFS is unknown, although there has been
evidence that viruses are environmental triggers. The authors found
that 67% of CSF patients are carriers of XMRV compared to 4% in a
healthy control population. The experimenters isolated the XMRV from
the patients and found that cell-associated and cell-free transmission
of the virus is possible. These results suggest that antivirals might
be effective treatment options for individuals suffering from CFS {2}.
Despite these advances, the cause of CFS is still unknown, as XMRV
could be acquired after CFS because CFS patients have weakened immune

References: {1} Pae et al. Expert Opin Pharmacother 2009, 10:1561-70
[PMID:19514866]. {2} Harmon K, "Retrovirus Linked to Chronic Fatigue
Syndrome, Could Aid in Diagnosis." Sci Am 2009, Oct 8 please click
here for article.

Competing interests: None declared
Evaluated 7 Dec 2009

David Wang
Washington University in St. Louis, United States of America

A recently identified retrovirus, xenotropic murine leukemia
virus-related virus (XMRV), has been detected more frequently in
patients with chronic fatigue syndrome than in healthy controls,
raising the possibility that there may be a disease association
. These
results underscore the fact that many diseases have poorly understood
etiologies and emphasize the need to identify and characterize
unrecognized pathogens.

XMRV was discovered initially in patients with prostate cancer {1}. In
this study, XMRV was detected in >60% of peripheral blood mononuclear
cells (PBMCs) of patients with chronic fatigue syndrome using a
polymerase chain reaction (PCR) assay. By contrast, <4% of controls
were PCR positive. A subset of the patient samples was analyzed for
the presence of XMRV antigen (by Western blot and
fluorescence-activated cell sorting [FACS]) and for infectious XMRV
virus (by co-culture experiments). These results support the notion
that XMRV is present in numerous patients with chronic fatigue
syndrome. Based on the PCR results of this study, there is a strong
association between XMRV and chronic fatigue syndrome.
It is clear
that larger scale case-control studies need to be performed (and there
will likely be many such studies appearing in the months to come) to
confirm these initial observations. Importantly, this study
illustrates that in this era of rapid identification of new microbes
in the human body, one must be mindful that a given microbe may have
pleiotropic effects (e.g. XMRV may be linked to both prostate cancer
and chronic fatigue or even additional disease states).

References: {1} Urisman et al. PLoS Pathog 2006, 2:25 [PMID:16609730].

Competing interests: None declared
Evaluated 18 Dec 2009

Ken Wilson
Duke University, United States of America

Chronic fatigue syndrome (CFS) is prevalent, highly debilitating and
frustrating for both patient and physician. A cause for the syndrome
may have been found at long last. Although etiology cannot at this
point be considered proven, Lombardi et al. present strong
circumstantial evidence that a retrovirus is involved.

CFS is a chronically debilitating syndrome with a prevalence of about
1%. Several aspects of the disease have suggested an infectious
etiology and various infectious agents have been implicated in the
past but causality has never been convincing. Lombardi et al. have
found that XMRV, a retrovirus closely related to murine leukemia
virus, can be detected in 2/3 of patients with CFS versus 3.7% of the
general population. Viral proteins were expressed in patients but not
controls and the patients had an immune response to viral proteins.
Both activated T-cells and activated B-cells were infected, and
infected cells taken from patients were able to transmit infection to
uninfected tissue culture cells. Although further work is clearly
required to prove or disprove a causal link, this paper probably
represents the strongest case so far for a specific etiologic agent in
this disorder.

Competing interests: None declared
Evaluated 7 Jan 2010

Charles Chiu
University of California, United States of America

This paper reports the detection of xenotropic murine leukemia
virus-related virus (XMRV) in blood from 67% of chronic fatigue
syndrome (CFS) patients but only 4% of controls in the United States,
a truly exceptional finding if confirmed
(some results have been
controversial). CFS is a devastating systemic, multi-symptom disease
that afflicts more than 17 million people worldwide without a defined
etiology to date.

Our group used a broad-spectrum viral microarray to detect XMRV in
patients with a specific kind of hereditary prostate cancer {1}.
Subsequently published data showed that XMRV was also detectable in
cases of sporadic prostate cancer {2}. This study is the first to look
for XMRV in peripheral blood mononuclear cells (PBMCs) of patients
with CFS using both nucleic acid (PCR) and antigen (Western blot,
FACS) methods. XMRV was detected in 67% of PBMCs from patients with
CFS but only in 4% of controls. This is a remarkable finding that now
associates XMRV with two widely different conditions: prostate cancer
and CFS, and whose data must be scrutinized carefully and confirmed
independently. In their Dissenting Opinion of this article, Moore and
Shuda raise valid concerns regarding the potential for PCR
contamination in this study. Some concerns include 1) the criteria for
defining CFS/ME in the patients and in controls were not explicitly
defined, 2) nested PCR was used and neither in a blinded nor
randomized fashion, 3) the remarkable lack of diversity in the six
fully sequenced XMRV genomes (<6 nucleotide average difference across
genome) -- with Fig. S1 even showing that for one fully sequenced
isolate two of the single nucleotide differences were "N's" -- clearly
the result of a sequencing error, 4) failure to use Southern blotting
to confirm PCR results, and 5) primary nested PCR screening done in
one lab as opposed to independent screening from start to finish in
two different laboratories. Concerns have also been brought up with
respect to the antigen testing. A subsequent publication in the UK has
refuted the results of this paper linking XMRV to cases of CFS {3},
but this can potentially be explained as representing geographical
differences in the condition, as XMRV was not seen in prostate cancer
in samples from European patients either {4}.

References: {1} Urisman et al. PLoS Pathog 2006, 2:e25
[PMID:16609730]. {2} Schlaberg et al. Proc Natl Acad Sci USA 2009,
106:16351-6 [PMID:19805305]. {3} Erlwin et al. PloS One, 5:e8519
[PMID:20066031]. {4} Hohn et al. Retrovirology 2009, 6:92

Competing interests: None declared
Evaluated 19 Jan 2010

Link to Dissenting Opinions & Author Responses on 'Detection of an
infectious retrovirus, XMRV, in blood cells of patients with chronic
fatigue syndrome'-

Dissenting Opinions & Author Responses on 'Detection of an infectious
retrovirus, XMRV, in blood cells of patients with chronic fatigue

Masahiro Shuda and
Patrick S Moore
University of Pittsburgh Cancer Institute, United States of America

The discovery of the cause of chronic fatigue syndrome would be an
extraordinary finding. Rather than providing extraordinary proof, this
manuscript has flaws that leave the reader unsure of knowing precisely
what was measured.

To detect the xenotropic murine leukemia-like virus (XMRV), the
authors used nested-PCR on non-randomized and non-blinded samples, a
recipe for uncontrolled PCR contamination. This technique re-amplifies
previously cycled products and is inherently prone to intermittent
false positivity that has occurred in our lab and many others (e.g.
{1} and {2} on which I am the author). This is a concern in light of
post-publication claims that XMRV detection rates among chronic
fatigue syndrome (CFS) patients have climbed from 67% to 95%, and XMRV
tests are now being sold and advertised on the internet at . Southern blotting, which would allay this
suspicion, was not done. Other results in the study also lack support.
Flow cytometry and immunostaining with murine leukemia virus (MLV)
antibodies were used to directly detect viral proteins in patient
cells (see Figure 2A of the paper). The CFS peripheral blood cells
have robust monotonic staining rather than the bimodal peaks that are
expected from a mixture of infected and uninfected populations of
peripheral blood cells. It is not certain whether this level of
viremia for an exogenous retrovirus is medically possible. It may,
perhaps, be possible but it seems improbable and is a pattern more
consistent with a cross-reactive endogenous retroviral antigen. To
confirm this finding, CFS peripheral blood cells (without negative
controls in Figure 2B) were immunoblotted using cross-reactive spleen
focus-forming virus (SFFV) and MLV antibodies. XMRV gp70 and p30
proteins are found at higher levels in 2 out of 5 CFS peripheral blood
samples (1150 and 1221) than in the positive control -- HCD-57 cells
directly infected with SFFV -- a very remarkable result. Repetition
with negative control samples (see Figure 2C of the paper) has the
higher molecular weight bands cut from the photograph, thus we cannot
interpret potential positivity for p30 gag precursor proteins among
the control samples (see CFS samples 1199 and 1220 in Figure 2B).
Finally, the positive control HCD-57 cell lane in Figure 2C lane 8 has
a completely different banding pattern from the very same control in
Figure 2B lane 7 for the p30 gag protein. The elementary issue of
whether the authors are measuring XMRV has to be clarified. The
fundamental basis for the CFS case and control samples is also not
defined at an appropriate level. The samples (supplementary online
material) were "selected for this study from patients fulfilling the
1994 CDC Fukuda Criteria for Chronic Fatigue Syndrome (S1) and the
2003 Canadian Consensus Criteria for Chronic Fatigue Syndrome/myalgic
encephalomyelitis (CFS/ME) and presenting with severe disability".
These are two separate definitions, the latter published in the
"Journal of Chronic Fatigue Syndrome" (which is no longer in print).
It is unclear how the samples were selected from these two criteria.
No references or cut-offs are given for tests used to clinically
define the CFS patients as cases so we are unable to interpret the
essential basis for the study. In addition, no description is given to
indicate that controls were tested in the same manner as CFS patients;
in fact, there is no description for negative control samples at all.
For a disease whose diagnosis is controversial, a clear statement of
where and how the cases and controls were selected is a critical first

Competing interests: None declared
Evaluated 18 Nov 2009

Author Response:

Judy Mikovits, Whittemore Peterson Institute, Reno, United States

This dissent first discusses "the cause of CFS". We did not imply that
XMRV caused CFS. We specifically state that our observation "raises
several important questions". Is XMRV infection a causal factor in the
pathogenesis of CFS or a passenger virus in the immunosuppressed CFS
The work presents a testable hypothesis that XMRV has a role
in CFS pathogenesis. The key task for the scientific community is to
define the scope of human disease associated with XMRV infection. We
were highly concerned and vigilant about taking precautions to avoid
PCR contamination. This was minimized using laboratory controls such
as dedicated first- and second-round PCR areas in different buildings
and consistent treatment of instruments and work areas with DNA ZAP
and UV rays. Although the original screen was performed using nested
PCR, PCR positivity on a subset of samples was confirmed using
single-round PCR in a different facility (Fig1A). Although not in the
manuscript, PCR status was verified on identical samples not processed
at WPI in a XMRV-free lab at NCI.
Moreover, in normal samples analyzed
in parallel, amplified sequences were found in only 3.7%, making it
unlikely that 67% positivity reflects "uncontrolled PCR
contamination". We should not have discussed unpublished data,
particularly considering the lay media. To discuss more details about
our more recent studies would repeat that error, but we were able to
culture XMRV virus from plasma and detect antibodies in some samples
which were PCR-negative (e.g. see #1118 in Fig1, 2A, 2D). These
examples compelled us to further study PCR-negative patient samples.
Flow cytometry data in the study were not meant to determine in vivo
levels of XMRV protein expression or the level of clinical viremia.
The goal was to determine if the XMRV DNA sequences detected
represented the presence of infectious viral particles. The studies of
XMRV protein expression in peripheral blood mononuclear cells (PBMC)
were performed after PBMC were activated in culture with
phytohemagglutinin and interleukin-2 for 7-14 days to allow the virus
to spread through the culture. It should have been made clearer.
Although at earlier times these samples did have bimodal peaks, data
shown were when most cells were virus positive. Not included in the
paper was the ability of azidothymidine to block XRMV spread in vitro.
The methodology for the immunoblots was also questioned. We used
monoclonal antibodies which recognized the envelope of all xenotropic
and polytropic but not ectopic murine leukemia viruses and reacted
with XMRV env proteins of expected sizes. Concerning the relatively
weak signal of the positive control, HCD-57/SFFV, test sample lanes
contained 150-200ug of protein, only 30ug was loaded in the HCD/SFFV
lane to prevent the positive control signal from overwhelming adjacent
lanes. This study was the initial finding of infectious XMRV virions
in human blood and a second association of XMRV and human disease. Our
observation of actively replicating virus, as well as antibodies
directed against XMRV (which were not criticized) in the patient
population examined, strengthened the paper and the hypothesis that
this recently discovered virus is a human pathogen. Clinically, CFS is
a heterogeneous syndrome with diagnosis made on the basis of the
exclusion of other diseases. Thus, the basis for diagnosis varies
greatly and we expect there to be XMRV-positive and -negative CFS
patients. Additional large-scale clinical studies using control groups
are essential to determine whether XMRV is the cause of CFS. Also,
rigorous independent validation is crucial. To facilitate this, the
NCI, Cleveland Clinic and WPI are making virus reagents available
through the NIH AIDS repository to any academic investigator by
contacting the investigators involved.

Response added 7 Jan 2010


Link to Faculty of 1000 Medicine comments on 'Detection of an
infectious retrovirus, XMRV, in blood cells of patients with chronic
fatigue syndrome'-

Faculty of 1000 Medicine comments on 'Detection of an infectious
retrovirus, XMRV, in blood cells of patients with chronic fatigue

Angélique van 't Wout and
Hanneke Schuitemaker
Academic Medical Centre, Amsterdam, Netherlands
Infectious Diseases

Xenotropic murine leukemia virus-related virus (XMRV), a novel
gammaretrovirus previously found in prostate tumor tissue, was found
significantly more often in the blood cells of chronic fatigue
syndrome (CFS) patients.

CFS is a disorder of unknown etiology that affects multiple organ
systems in the body, including chronic activation of the innate immune
system and a deficiency in natural killer (NK) cell activity. A number
of viruses, including ubiquitous herpesviruses and enteroviruses have
been implicated as possible environmental triggers of CFS. Previously,
XMRV was found in the tumor tissue of a subset of prostate cancer
patients. Since prostate cancer and CFS have both been linked to
alterations in the antiviral enzyme ribonuclease (RNase) L, the aim of
the authors was to test the association of XMRV with CFS
. The authors
indeed found XMRV sequences and protein expression in peripheral blood
mononuclear cell (PBMC) DNA from CFS patients. Moreover, they showed
the presence of infectious XMRV in PBMCs and plasma from CFS patients
as well as plasma XMRV antibody reactivity. The low frequency and/or
absence of each of these XMRV signals in control patients suggest an
association between XMRV and CFS. However, Koch's postulates remain to
be fulfilled and many questions remain -- e.g. is XMRV cause or
consequence? What is the pathogenic mechanism? Both epidemiological
and laboratory studies will need to confirm and expand on the observed
association between XMRV and CFS.

Competing interests: No potential interests relevant to this article
were reported.
Evaluated 28 Oct 2009


Link to Faculty of 1000 Biology comments on 'Host range and cellular
tropism of the human exogenous gammaretrovirus XMRV', Stieler K et al.

Faculty of 1000 Biology comments on 'Host range and cellular tropism
of the human exogenous gammaretrovirus XMRV', Stieler K et al. 2010

David Alpers
Washington University School of Medicine, United States of America

A viral cause for chronic fatigue syndrome (CFS), often co-morbid with
irritable bowel syndrome (IBS), has been suggested before. A pair of
recent reports has suggested that xenotropic murine leukemia
retrovirus (XMRV) is/is not associated with CFS, and the fascinating
study reviewed here examines the factors that permit and regulate
viral expression in mammalian cells.

XMRV was first associated with prostatic cancer samples in North
America, but the virus was rarely found in samples from Northern
Europe {1,2}. Lombardi et al. reported XMRV in nearly 70% of
peripheral blood cells from patients with CFS in North America {3},
but Erlwein et al. could not confirm this in a UK sample {4}. Stieler
et al. show that the murine lymphotropic virus (MLV) receptor, XPR1,
mediates XMRV uptake but that receptor number could not solely account
for variations in titer in cells. Moreover, some cells could be
infected but not produce complete secreted virus. Whether factors
affecting replication, integration, assembly or release can account
for the reported differences in prevalence in prostatic tumor tissue
in North America and Northern Europe is not yet clear. CFS is a
disabling syndrome, and, if it were shown to be due to an infectious
agent, could revolutionize the approach to many functional co-morbid
syndromes, including IBS. But the original reports that human T cell
lymphotropic virus (HTLV)-type II was associated with over 80% of CFS
cases were not confirmed, nor could other retroviruses be found
associated {4}. XMRV is an exogenous gammaretrovirus, and it is not
clear if the disagreement already reported for CFS is related to
difficulties in demonstrating retroviral presence or to some other
issues. Further studies are needed and based on these early reports
will follow rapidly in numbers large enough to provide the needed

References: {1} Fischer et al. J Clin Virol 2008, 43:277-83
[PMID:18823818]. {2} Hohn et al. Retrovirology 2009, 6:92
[PMID:19835577]. {3} Lombardi et al. Science 2009, 326:585-9
[PMID:19815723]. {4} Erlwein et al. PLoS One 2010, 5:e8519

Competing interests: None declared
Evaluated 3 Feb 2010

1 comment:

oerganix said...

XMRV has been found in UK, so let's put to rest the idea that it is regional. It has also been found in Japan. The WPI study included samples from UK, Ireland, Germany and Australia. Two groups of patients living in UK also had their blood tested using WPI tests and approximately 50% tested positive.

The medical establishment in UK decided long ago that ME/CFS is a mental illness and therefore patients are not allowed to be tested for organic illness. The politics behind this ridiculous assumption were working in the UK study and they came up with the results they wanted, to support their bias. Any study with Dr Wessely's name on it should be looked at with suspicion as he characterizes ME/CFS patients as malingerers suffering from 'illness beliefs' - not exactly a guy looking for scientific answers.