Failure to Detect the Novel Retrovirus XMRV in Chronic
Otto Erlwein1, Steve Kaye1, Myra O. McClure1*, Jonathan Weber1, Gillian Wills1, David Collier2, Simon
Wessely3, Anthony Cleare3
1Jefferiss Research Trust Laboratories, Section of Infectious Diseases, Wright-Fleming Institute, Faculty of Medicine, Imperial College London, St Mary’s Campus, Norfolk
Place, London, United Kingdom, 2Social Genetic and Developmental Psychiatry Centre, Institute of Psychiatry (King’s College London) De Crespigny Park, Denmark Hill,
London, United Kingdom, 3Department of Psychological Medicine, Institute of Psychiatry, King’s College London, Camberwell, London, United Kingdom
with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate
cancer.Thisfinding,if confirmed, would haveaprofound effect on the understanding and treatment ofanincapacitating disease
affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV.
Methodology: Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met
the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for
the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity
of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious
molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV
sequences were detected.
Conclusion: XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we
found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North
America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US
groups found XMRV in prostate cancer tissue, while two European studies did not.
Citation: Erlwein O, Kaye S, McClure MO, Weber J, Wills G, et al. (2010) Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome. PLoS ONE 5(1):
Editor: Douglas F. Nixon, University of California San Francisco, United States of America
Received December 1, 2009; Accepted December 4, 2009; Published January 6, 2010
Copyright: ? 2010 Erlwein et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: AC, DC and SW are partially funded by the South London and Maudsley NHS Foundation Trust/Institute of Psychiatry National Institute of Health
Biomedical Resaerch Centre. The team from Imperial College is grateful for support from the NIHR Biomedical Research Centre Funding Scheme. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
A recent study by Lombardi et al.  describing a gamma-
retrovirus infection in 68 of 101 chronic fatigue syndrome (CFS)
patients was notable not only for its claim of a new viral aetiology of
a hitherto controversial disease, but also for the fact that proviral
DNA could be amplified from the peripheral blood mononuclear
cells (PBMC) of 3.75% (8/218) of the healthy controls. This follows
an earlier claim that 1.7% (5/300) of healthy Japanese blood donors
carried antibodies to the same virus . The virus in question is a
recently discovered retrovirus, Xenotropic Murine Leukaemia
Virus (MLV)-Related Virus (XMRV).
In the original identification of XMRV in prostate cancer
stromal cells, Urisman et al.  confirmed by sequence analysis
that XMRV is not a laboratory contaminant, as is often the case
with claims of new retroviral associations with disease. It shares
.90% sequence identity in gag and env (two of the three viral
structural genes) with other xenotropic MLVs.
An association between XMRV and prostate cancer was
strengthened with the demonstration of XMRV protein expres-
sion in malignant epithelial cells . However, these results have
not been duplicated in studies conducted in Europe [5–7]. Both
prostate cancer and CFS have been linked to an Arg to Gln
mutation at codon 462 (R462Q) in the RNaseL gene, an
interferon-induced ribonuclease . On activation, RNaseL
destroys single stranded cellular and viral RNA, thereby
preventing viral replication, blocking protein synthesis, triggering
cellular apoptosis and providing an innate anti-viral response.
The two US studies are of interest, not only because this would be
a further example of a virus association with cancer, but because
they represent the first demonstration of a gamma-retrovirus able
toinfect human cells,over-riding
mechanisms that were believed to protect humans from MLV
The XMRV sequences derived from prostate cancer tissue are
identical to those from CFS patients, but differ from xenotropic
MLV sequences, endorsing a genuine cross-species transmission.
However, the claim that XMRV is preferentially found in prostate
tumours from patients homozygous for the R462Q variant  is
not borne out by the second prostate cancer study to find XMRV
in patients , nor was the genetic variant detected in CFS
patients carrying XMRV .
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The finding of Lombardi et al. of a 67% XMRV infection rate
among CFS patients, if confirmed, would have a serious impact on
understanding the pathogenesis of this complex and debilitating
disease and its treatment. Therefore, it was important to determine
if CFS sufferers in the UK were carriers of XMRV. We have
screened DNA extracts from the blood of CFS sufferers by PCRs
targeted at an XMRV-specific sequence and at a sequence
conserved amongst most murine retroviruses (MRV).
All patients gave written informed consent for the use of their
DNA to test aetiological theories of CFS, and the study was
approved by the South London and Maudsley NHS Trust Ethics
Committee. The study recruited 186 patients (62% female, age
range 19–70, mean 39.6611.3years) from consecutive referrals to
the CFS clinic at King’s College Hospital, London. All patients
had undergone medical screening to exclude detectable organic
illness, including a minimum of physical examination, urinalysis,
full blood count, urea and electrolytes, thyroid function tests, liver
function tests, 9 a.m. cortisol and ESR. Patients were interviewed
using a semi-structured interview for CFS  to determine
whether they met international consensus criteria for CFS. All
subjects met the CDC criteria ; patients with the Fukuda-
specified exclusionary psychiatric disorders, or somatisation
disorder (as per DSM-IV), were not included. The patient set
studied is a well-characterised and representative sample of CFS
patients who have been described previously: all were routine
clinic attendees, referred within the UK National Health Service,
who had taken part in prior studies of neuroendocrine functioning
 and/or of cognitive behaviour therapy . As is typical of
the patients seen in this tertiary care centre, they were markedly
unwell. Few were working, and 19% were members of patient
support groups for CFS/ME [12–14]. The levels of fatigue in this
sample were high (mean Chalder Fatigue Scale, 26.365.4) , as
were levels of disability (mean Work and Social Adjustment Scale,
total score 28.267.2) . The mean GHQ-12 score  was
19.768.1. Patients had been unwell for a median of 4.0 y (range
1–28 y). Of note was that 45% said their illness definitely related
to a viral illness and 45% said it might relate to a viral illness.
Overall, we conclude that this sample is typical of CFS patients
seen in specialist clinical services in the UK. We also know from
collaborative studies that our patients resemble those seen in other
specialist CFS services in the United States and Australia .
PCR detection of XMRV and MLV sequences.
extracted from EDTA whole blood using a standard phenol-based
organic deproteinisation procedure . DNA concentrations
were determined by absorbance at 260 nm (A260). Each sample
was amplified in three nested PCRs using primers targeted to an
XMRV-specific sequence, to a sequence conserved amongst most
MLV and, as a control for sample addition and PCR-inhibition, to
a human beta-globin (hBG) sequence (Table 1). Each first-round
reaction was performed in a 25 ml volume containing 0.5 units
TaqGold (Applied BioSystems, Warrington, UK), 1 x TaqGold
reaction buffer (Applied BioSystems), 1.5 mM Mg2+, 200 mM
each dNTP, 2.5 pmol each primer to which 5 ml DNA extract or
control was added. Reaction conditions were one cycle of 94uC, 8
minutes, 35 cycles of 94uC 30 seconds, 55uC 30 seconds, 72uC 30
seconds and one cycle 0f 72uC, 7 minutes. Second round reaction
mixes were identical to the first round and the sample was a 1 ml
transfer from the first round reactions. Second round reaction
conditions were as for the first round over 30 cycles. PCR
amplicons were visualised on a 1% agarose gel stained with
ethidium bromide. Each PCR run consisted of test samples, six
negative (water) and two positive controls. The positive control
was a dilution of a plasmid with a full-length XMRV (isolate
VP62) insert, generously gifted by Dr R. Silverman. To validate
the sensitivity of the PCR, an end-point dilution of the plasmid was
performed. To determine specificity of the PCR, a sample of
human DNA from the LNCaP prostate cancer cell line (American
Type Culture Collection, code CRL-1740) was amplified with the
XMRV and MLV primer sets. To ensure integrity of the DNA
extracts, three randomly selected samples were titrated to end-
point using the hBG PCR to determine if the PCR copy number
equated with the A260. To determine if the DNA extracts exhibited
low level non-specific inhibition of PCR, 10 samples were
subjected to 30 cycles of the first round hBG PCR (reaction mix
and conditions as above) followed by 40 cycles of a nested real-
time SYBR-green PCR using the SYBR-green Fast PCR kit
(Roche, Lewes UK) according to the manufacturer’s instructions.
Nested PCR Validation
Based on A260 of the purified plasmid, both primer sets
(XMRV, MLV) were able to amplify a single target copy added to
the reaction. Amplification of 600 ng of LNCaP cellular DNA
added to XMRV and MLV PCRs yielded no non-specific bands
when viewed on an ethidium bromide-stained agarose gel.
Quantification of DNA samples from three randomly selected
test samples by end-point dilution PCR with the hBG primer set
showed concurrence of the PCR-determined copy number with
A260, thus indicating integrity of the DNA preparations. Nested
real-time amplification of 10 samples showed no evidence of non-
specific inhibition as determined by the slope of the amplification
curves and the height of the signal plateau.
PCR Analysis of Test Samples
Input DNA ranged from 10 to 600 ng (1.66103to 1.16105cell
equivalents) as determined by A260of which 149 samples had an
input of .100 ng and 106 samples .200 ng. None of the 186 test
samples analysed yielded a specific PCR product with either the
XMRV or MLV primer sets and no non-specificPCR products were
observed. A specific hBG product was amplified from all 186 test
samples. The positive control was amplified in each run by the
Table 1. Oligonucleotide Primers.
Target Sequence Location
XMRVForward outer 59CATTCTGTATCAGTTAACCTAC 39
Reverse outer 59 ATGATCTCGAGAACACTTAAAG 39
Forward inner 59 GACTTTTTGGAGTGGCTTTGT 39
Reverse inner 59 ACAGAAGAACAACAAAACAAATC 39
MLV Forward outer 59 GGATCAAGCCCCACATACAG 39
Reverse outer 59 CATCAAACAGGGTGGGACTG 39
Forward inner 59 AGAAGTCAACAAGCGGGTGG 39
Reverse inner 59 GGTGGAGTCTCAGGCAGAAA 39
hBGForward outer 59 TGGTGGTCTACCCTTGGACC 39
Reverse outer 59 GAGGTTGTCCAGGTGAGCCA 39
Forward inner 59 GAGGTTCTTTGAGTCCTTTGG 39
Reverse inner 59 CATCACTAAAGGCACCGAGCA 39
Locations in GenBank accessions1EF185282,2NM000518.4.
XMRV and CFS
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PCR products is shown in figure 1 and a representativesampleof our
results with CFS DNA and MLV primers is shown in figure 2.
Unlike the study of Lombardi et al., we have failed to detect
XMRV or closely related MRV proviral DNA sequences in any
sample from CFS cases. There have been numerous claims for an
infective aetiology to CFSover the years, not least because, as in this
sample, many patients report that their symptoms were triggered by
an infective episode. Prospective epidemiological studies have
confirmed that certain infective agents, for example Epstein Barr
virus, are unequivocally associated with subsequent CFS , even
if the mechanisms are unclear and almost certainly multi factorial.
Nearly two decades ago, sequences from another retrovirus, the
human T-lymphotropic virus type ll, were amplified from the
PBMCs of 10/12 (83%) adult and 13/18 paediatric CFS patients,
but not from healthy control subjects . However, subsequent
studies carried out on small numbers (20–30) of CFS patients, failed
to confirm evidence for HTLV (type 1 or 11) [22–25] or other
retroviruses, including the closely-related simian T lymphotropic
feline leukaemia viruses  and HIV-1 .
The Lombardi paper is the first to study a significantly larger
number of people than that in any previous study and to detect a
virus only recently discovered. Our study resembles that of
Lombardi et al. in certain respects. Both studies use the widely
accepted 1994 clinical case definition of CFS10. Lombardi et al.
reported that their cases ‘‘presented with severe disability’’ and we
provide quantifiable evidence confirming high levels of disability in
our subjects. Our subjects were also typical of those seen in
secondary and tertiary care in other centres.
Our own study also differs from that of Lombardi in other
respects. Firstly, the PCR operator was blinded to the provenance
of the DNA samples. In fact, with the exception of the PCR
controls, all 186 DNA test samples originated from CFS patients.
Care was taken to grow the XMRV plasmid in a laboratory in
which no MLV had been cultured and no MLV vectors used and
the PCR was carried out in a CPA-accredited Molecular
Diagnostics Unit which processes only human tissue. Multiple
(six) water (negative) controls were included in every run to detect
low level contamination and a PCR to amplify a sequence that is
conserved in most murine leukaemia viruses was included in order
to expose any circulating MLV contamination and to detect any
variant of XMRV that might be circulating in the UK CFS
Based on our molecular data, we do not share the conviction
that XMRV may be a contributory factor in the pathogenesis of
CFS, at least in the U.K.
The assistance of Sarah Bull in data collection and processing is gratefully
Conceived and designed the experiments: SK MM. Performed the
experiments: OWE SK. Analyzed the data: SK MM. Contributed
reagents/materials/analysis tools: SK GW DC SW AC. Wrote the paper:
SK MM. Facilitated the study by setting up the collaboration: JW.
Responsible for providing samples and associated data from a well
characterised and valuable cohort of subjects: SW.
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Figure 1. PCR products of the XMRV VP62 clone. Primers are
generic to MLV (lanes 1 and 2) or specific to XMRV (lanes 4 and 5). The
sizes of the respective fragments are shown. Lane 3–200 bp molecular
Figure 2. Nested PCR from the DNA of 8 CFS patients. Products
of generic MLV primers (including XMRV) are shown. Lanes 1–8, CFS
patient DNA (2ndround); lanes 9 and 10, XMRV 2ndround and 1stround
positive controls; lanes 11 and 12, DNA of uninfected cell line LNCaP;
lanes 13–18, water controls.
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