/ www.sciencexpress.org / 31 May 2011 / Page 1/ 10.1126/science.1204963
Murine-like gammaretroviruses (MLVs), most
notably XMRV [xenotropic murine leukemia virus
(X-MLV)–related virus], have been reported to be
present in the blood of patients with chronic fatigue
syndrome (CFS). We evaluated blood samples from
61 patients with CFS from a single clinical practice,
43 of whom had previously been identified as
XMRV-positive. Our analysis included polymerase
chain reaction and reverse transcription polymerase
chain reaction procedures for detection of viral
nucleic acids and assays for detection of infectious
virus and virus-specific antibodies. We found no
evidence of XMRV or other MLVs in these blood
samples. In addition, we found that these
gammaretroviruses were strongly (X-MLV) or
partially (XMRV) susceptible to inactivation by sera
from CFS patients and healthy controls, which
suggested that establishment of a successful MLV
infection in humans would be unlikely. Consistent
with previous reports, we detected MLV sequences
in commercial laboratory reagents. Our results
indicate that previous evidence linking XMRV and
MLVs to CFS is likely attributable to laboratory
Xenotropic retroviruses, first discovered in mice, have
the unusual characteristic of being endogenous to
animal species, i.e., integrated into the animal’s
genome, but not able to re-infect cells from that species.
However, as the name (xenos, foreign) implies, these
viruses can infect cells from other animal species. The
xenotropic murine leukemia virus (X-MLV), for
example, infects cells from several species including
humans but cannot infect many mouse cells (1–3). One
particular virus within this group, XMRV (xenotropic
murine leukemia virus-related virus), has been reported
to be present in a subset of human prostate tumors (4)
and in blood samples from patients with chronic fatigue
syndrome (CFS) (5). Other murine-related
gammaretroviruses (MLVs) have also reportedly been
detected in CFS patients (6). The infection of humans
with these viruses is controversial. Investigators
evaluating independent cohorts of CFS patients have
failed to detect XMRV or other MLV (7–12), and
contamination of human clinical material (13, 14) and
reagents (e.g., Taq polymerase) (15) with mouse DNA
containing MLV-like sequences has been reported.
To investigate these discrepancies in a more direct
manner, we performed an extensive virological
evaluation of blood samples from two human
populations with a clinical diagnosis of CFS (16), many
of whom had been diagnosed previously as XMRV-
infected. The first (P1) consisted of 41 CFS patients
ranging in age from 5 years to 73 years who came from
a private medical practice (Sierra Internal Medicine,
Incline Village Nevada). Twenty-six of the CFS
subjects (63%) were female and 15 (37%) were male;
the female median age was 52 years (range 5 to 72
years) and male median age was 49 years (range 20 to
73 years). These patients were an unselected,
sequentially enrolled population submitted for
diagnostic testing to Wisconsin Viral Research Group
(WVRG), and were therefore a true cross-section of the
patients in the medical practice. Thirty-seven of these
41 patients had been tested previously for XMRV
infection by the following assays: whole blood PCR,
serum PCR, or viral XMRV culture with PCR (17).
No Evidence of Murine-Like Gammaretroviruses in CFS Patients Previously
Identified as XMRV-Infected
Konstance Knox,1,2 Donald Carrigan,1,2 Graham Simmons,3,4 Fernando Teque,5 Yanchen Zhou,3,4 John Hackett Jr.,6
Xiaoxing Qiu,6 Ka-Cheung Luk,6 Gerald Schochetman,6 Allyn Knox,1 Andreas M. Kogelnik,2 Jay A. Levy5*
1Wisconsin Viral Research Group, Milwaukee, WI 53226, USA. 2Open Medicine Institute, Mountain View, CA
94040, USA. 3Blood Systems Research Institute, San Francisco, CA 94118, USA. 4Department of Laboratory
Medicine, University of California, San Francisco, San Francisco, CA 94143, USA. 5Department of Medicine,
Hematology/Oncology Division, University of California, San Francisco, San Francisco, CA 94143, USA. 6Abbott
Laboratories, Abbott Park, IL 60064, USA.
*To whom correspondence should be addressed. E-mail: Jay.Levy@ucsf.edu
on May 31, 2011
/ www.sciencexpress.org / 31 May 2011 / Page 2/ 10.1126/science.1204963
These evaluations were performed by a commercial
(VIPDx, Reno, NV) or research laboratory (Whittemore
Peterson Institute [WPI], Reno, NV). Twenty-six were
reported as being XMRV positive and 11 were reported
as being negative. Blood samples used from this patient
cohort were archived diagnostic specimens and,
therefore, exempt from IRB consideration [46.101
(b)(4), Code of Federal Regulations].
The second population (P2) came from the same
medical practice and subjects were selected largely on
the basis of a previous positive diagnosis for XMRV
infection. This patient cohort included 29 CFS patients,
26 of whom (89.6%) had tested positive for XMRV in
at least one of the three virus assays listed above, and/or
had antibodies to XMRV detected in a commercial
(VIPDx) or research laboratory (WPI) (5) (table S1).
Twenty of the patients (69%) were female and 9 (31%)
were male with a median age of 52 years. Nine of these
subjects were also part of P1 (table S1). Fresh blood
samples were used for viral culture and testing (see
supporting online material). For the serum inactivation
studies, seven healthy UCSF laboratory workers,
ranging in age from 21 to 72 years, served as controls.
These volunteers were afebrile without signs of any
illness. This research received approval of the Human
Subjects Committee at the University of California, San
Francisco. All participants signed IRB-approved
We initially assessed the peripheral blood leukocytes
from the 41 subjects in P1 for XMRV DNA using
nested PCR targeting gag (primers 419F/1154R and
445F/870R) and env (primers 5922F/6273R and
5937F/6198R). The sensitivity of these PCR assays is at
least 10 XMRV genomes per reaction (table S3). No
XMRV DNA was detected in any sample (see Fig. 1A
for representative data). Notably, a chart review of the
41 patients revealed that 19 had two blood samples
drawn on the same day by the same phlebotomist, with
one sample submitted to VIPDx and the other to
WVRG. For XMRV analysis, VIPDx used diagnostic
technologies identical to those utilized in previous
studies on XMRV and CFS (5). The chart review
indicated that 53% (10/19) of the blood samples were
reported by the commercial laboratory as being positive
for XMRV DNA. This difference in our results (0/19)
versus the chart review results (10/19) was statistically
significant (p< 0.0004, two-sided Fisher's Exact test).
Our failure to detect XMRV DNA in patient
population P1 prompted us to undertake a more
extensive study of patient population P2. We used
multiple methodologies to evaluate P2 blood samples
for the presence of (i) nucleic acids derived from
XMRV or MLV; (ii) infectious XMRV and MLV; and
(iii) XMRV-specific antibodies (17). Ficoll-Hypaque
purified peripheral blood mononuclear cells (PBMC)
were evaluated by RT– PCR procedures directly or
after activation with phytohemagglutinin (PHA; 3
μg/ml for 3 days) using primers and protocols described
by others (6) and previously demonstrated to be highly
sensitive for detection of XMRV and MLVs (6, 18). In
addition, plasma was evaluated by RT-PCR in a similar
manner. No MLV was found in the PBMC or plasma of
these 29 CFS patients (Table 1, Fig. 1B). The positive
control, consisting of the 730 bp fragment of XMRV
amplified from prostate cancer cell line, 22Rvl, was
able to detect at least 10 copies of XMRV gag DNA per
reaction; second-round PCR detected 1-10
copies/reaction (table S3).
We next investigated whether infectious XMRV or
MLV was detectable in the P2 blood samples. The
patients’ PBMCs were added to duplicate plates of
early-passaged mink lung cells to enhance detection of
X-MLV and maintained for 5 days (2, 19, 20). The
PBMCs were then removed and the mink lung cells
passed weekly for 3 weeks. Culture fluids were then
evaluated for infectious XMRV or MLV by monitoring
the induction of focus formation in the mink S+L- cell
line (19, 20), by measuring RT activity in the cell
culture fluids (21), and by PCR analysis (11, 18). We
also looked for infectious virus in culture fluids from 19
patient PBMCs that had been cultured for 1-3 weeks
after PHA stimulation. As summarized in Table 1, we
did not detect XMRV or MLV in any of the patient
A previous study reported that 50% (9/18) of
patients with CFS had plasma antibodies reactive with
XMRV (5). We evaluated 60 plasma samples from P1
and P2 patients for the presence of XMRV-specific
antibodies by means of two direct format
chemiluminescence immunoassays (CMIAs) using
either transmembrane p15E or envelope gp70
recombinant proteins of XMRV (22). These assays can
detect antibodies to other MLVs. None of the 60 plasma
samples from these CFS patients was reactive in the
p15E CMIA (Fig. 2A). One of the 60 samples was
weakly reactive in the gp70 CMIA with a sample/cut-
off (S/CO) value of 5.4 (Log N of S/CO = 1.68).
However, the plasma was not positive by Western blot
on May 31, 2011
/ www.sciencexpress.org / 31 May 2011 / Page 3/ 10.1126/science.1204963
(WB) assay using purified XMRV viral lysate as well
as recombinant gp70 protein (22) (Fig.2B). It was
therefore considered negative.
Further studies of antiviral responses in the P2
population assessed whether serum samples from these
patients could inactivate X-MLV and XMRV. Previous
work (23) had indicated that X-MLV is sensitive to
inactivation by sera from healthy individuals, most
likely by human complement (24–26); conceivably,
CFS patient sera are deficient in this activity. X-MLV
and XMRV were mixed with unheated or heated human
sera from 7 healthy subjects and from 19 CFS patients
(17). Both viruses were susceptible to inactivation by
unheated, complement-containing sera from both
groups; over a 2-log reduction in virus infectivity was
noted in several cases. XMRV was less susceptible to
inactivation than X-MLV (Fig. 3) most likely reflecting
the passage of XMRV through human cells, which
renders the virus less sensitive to human complement
(24–26). These results, as well as other reports showing
restriction of XMRV replication in human cells (27,
28), suggest that an established MLV infection in
humans is unlikely.
Because neither XMRV or MLV sequences or
infectious virus could be detected in the blood of the 61
CFS patients in our P1 and P2 populations, we explored
whether XMRV and MLV sequences might be present
in research reagents used to detect these viruses. While
our own studies were underway, other investigators
considered the same possibility (29) and reported that
mouse DNA and MLV sequences were detectable in
reagents and tissues used for RT-PCR (13–15),
particularly the mouse monoclonal antibodies (MAbs)
in Taq polymerase preparations (15). Notably, we
detected MLV sequences not only in 3/5 Taq
polymerases that utilize MAbs, but also in 9/17 other
MAbs-containing reagents used in research laboratories
(table S2) including antibodies to CD4, CD8, and
CD14. Sequencing of these PCR products revealed a
high degree of sequence homology with known MLV
sequences from laboratory strains; they most closely
resembled the MLV sequences reported by others in the
blood of CFS patients (6) (figs. S1 and S2).
Bioreagent contamination, however, does not
adequately explain the detection of XMRV by
Lombardi et. al. (5). We have found that the DNA
sequences of 3 XMRV proviruses they described are
identical to that of VP62, which is the prototype XMRV
cloned from prostate cancer tissue (4). Long-term
passage of VP62 led to proviruses with accumulated
multiple point mutations (fig. S3). As suggested by
others (30), independently derived XMRV DNA
sequences should show increased genetic diversity
compared to the VP62 clone sequence. Therefore, the
remarkable conservation of the WPI-XRMV sequences
is most consistent with laboratory contamination with
the original infectious VP62.
In conclusion, we have found no evidence that
XMRV or other murine-like gammaretroviruses are
present in blood samples from 43 CFS patients who
were previously reported to be infected by XMRV (5,
6). Notably, over a period of several months, 7 of our
subjects were studied on two occasions; 2 subjects, on
three occasions. Because our blood samples were
obtained from CFS patients from the same clinical
practice that provided the majority of patients described
in the early XMRV report (5), differences in the patient
cohort or clinical diagnosis cannot account for the
discrepancies between our findings and the previous
observations. We believe that the detection of MLV in
human blood in previous studies (5, 6) reflects
contamination of reagents used to assess their presence
and/or contamination of human samples during
laboratory manipulation of the infectious XMRV clone,
VP62 (5). In addition, our studies indicate that X-MLV
and XMRV are fully or partially inactivated by human
serum, suggesting that these viruses could not readily
establish a human infection. Since an activated immune
system has been observed in CFS patients (31), the
possibility of another infectious agent(s) being
associated with this illness merits continual attention.
1. J. A. Levy, Science 182, 1151 (1973).
2. J. A. Levy, Current Topics in Microbiology and
Immunology 111 (1978).
3. S. Baliji, Q. Liu, C. A. Kozak, J Virol 84, 12841
4. A. Urisman et al., PLoS Pathog 2, e25 (2006).
5. V. C. Lombardi et al., Science 326, 585 (2009).
6. S. C. Lo et al., Proc Natl Acad Sci U S A 107, 15874
7. O. Erlwein et al., PLoS One 5, e8519 (2010).
8. T. J. Henrich et al., J Infect Dis 202, 1478 (2010).
9. H. C. Groom et al., Retrovirology 7, 10 (2010).
10. P. Hong, J. Li, Y. Li, Virol J 7, 224 (2010).
11. W. M. Switzer et al., Retrovirology 7, 57 (2010).
12. F. J. van Kuppeveld et al., Bmj 340, c1018 (2010).
13. B. Oakes et al., Retrovirology 7, 109 (2010).
References and Notes
on May 31, 2011
/ www.sciencexpress.org / 31 May 2011 / Page 4/ 10.1126/science.1204963
14. M. J. Robinson et al., Retrovirology 7, 108 (2010).
15. E. Sato, R. A. Furuta, T. Miyazawa, Retrovirology
7, 110 (2010).
16. K. Fukuda et al., Annals of Internal Medicine 121,
17. Materials and methods are available as supporting
material on Science Online.
18. G. Simmons, e. al., Transfusion 51, 643 (2011).
19. O. E. Varnier, A. D. Hoffman, B. A. Nexo, J. A.
Levy, Virology 132, 79 (1984).
20. O. E. Varnier, C. M. Repetto, S. P. Raffanti, A.
Alama, J. A. Levy, J Gen Virol 64, 425 (1983).
21. A. D. Hoffman, B. Banapour, J. A. Levy, Virology
147, 326 (1985).
22. X. Qiu et al., Retrovirology 7, 68 (2010).
23. B. Banapour, J. Sernatinger, J. A. Levy, Virology
152, 268 (1986).
24. Y. Takeuchi et al., J Virol 68, 8001 (1994).
25. R. P. Rother et al., J Exp Med 182, 1345 (1995).
26. D. M. Takefman, G. T. Spear, M. Saifuddin, C. A.
Wilson, J Virol 76, 1999 (2002).
27. T. Paprotka et al., J Virol 84, 5719 (2010).
28. H. C. Groom, M. W. Yap, R. P. Galao, S. J. Neil, K.
N. Bishop, Proc Natl Acad Sci U S A 107, 5166
29. R. Weiss, BMC Biology 8, 124 (2010).
30. S. Hue et al., Retrovirology 7, 111 (2010).
31. A. L. Landay, C. Jessop, E. T. Lennette, J. A. Levy,
Lancet 338, 707 (1991).
Acknowledgments: We thank D. Peterson for referral
of all the subjects evaluated; J. Weismann for
coordinating subject participation; I. Livinti for
assistance with data management; E. Delwart and J.
Fiddes for comments on the manuscript; and K.
Peter for help in preparation of the manuscript.
These studies were conducted with support of
private funds to the investigators. Patent applications
have been filed by Abbott Laboratories relating to
detection of XMRV using immunoassays and
Supporting Online Material
Materials and Methods
Figs. S1 to S3
Tables S1 to S3
1 March 2011; accepted 16 May 2011
Published online 31 May 2011; 10.1126/science.1204963
Fig. 1. (A) Representative nested gag PCR results using
genomic DNA (gDNA) from P1 patient leukocytes. A
negative control (water, lane 0) and two positive
controls (XMRV gag plasmid at 10 and 100
copies/reaction) were included in each run. As control,
patient DNA was also tested with single-round PCR for
RNaseL (17). DNA markers (M) and the positions of
expected PCR products are annotated. (B)
Representative nested RT-PCR results on P2 PBMC
samples. Positive and negative controls are shown. Ten-
fold serial dilutions of XMRV gag plasmid control start
at 1000 copies/reaction. Negative controls for each
reaction step were tested in triplicate: *RNA/DNA
extraction negative control, **RT control, and ***PCR
Fig. 2. Evaluation of 60 CFS plasma samples for the
presence of XMRV antibodies. (A) Two recombinant
protein-based CMIAs were used to detect specific
antibodies to XMRV gp70 and p15E proteins (17). The
X axis represents the CMIA signal in a unit of natural
log-transformed ratio of sample signal to the cutoff
signal (Log N S/CO). (B) Western blot analysis of gp70
CMIA reactive CFS sample using native XMRV viral
proteins and mammalian-expressed recombinant gp70
protein. Sample keys: the gp70 CMIA-reactive (CFS)
sample 09-7571, Positive control (PC) of antisera of
XMRV-infected macaque, negative control (NC) of
normal blood donor and Molecular weight (MW)
markers in kilodaltons (KD).
Fig. 3. Effects of Human Serum on Xenotropic MLV
and XMRV. Shown is the percent serum inactivation of
virus, as measured by induction of focus formation in
mink S+L- cells by untreated X-MLV and XMRV (17).
Representative results are shown. Unheated sera from
12 other CFS patients gave similar findings with nearly
complete inactivation of X-MLV and partial to high
inactivation of XMRV. X-MLV was obtained from
NZB mouse cells and propagated in mink lung cells
(20). XMRV was obtained from the human prostate cell
line (22Rv1). For the five studies conducted, the control
virus titers measured as focus formation in mink S+L-
cells were 126, 430, 168, 246, 208 foci (X-MLV); 84,
376, 208, 284, 206 foci (XMRV). N, control; P, CFS
patient (see table S1); black bars, X-MLV unheated
sera; shaded bars, X-MLV heated sera; white bars,
XMRV unheated sera; hatched bars, XMRV heated
on May 31, 2011
/ www.sciencexpress.org / 31 May 2011 / Page 5/ 10.1126/science.1204963
Table 1. Summary of assays used to evaluate blood samples from CFS patients in population P2. Information
about the CFS patients is provided in table S1. Two subjects were studied twice within a 3-month period (table S1)
and gave the same results.
PCR analysis of PBMC-derived DNA
RT-PCR analysis of patient plasma
PBMC culture fluids*
Reverse transcriptase assay of supernatants from
mink lung cells passed after PBMC co-culture*
*Infectious virus assay: Fluids were tested for infectious virus production by reverse transcriptase (RT) and the
mink S+L- cell assays (see text) (17). †Insufficient cells were available for these studies from subject #24.
Percent XMRV-positive (n)
on May 31, 2011
Page 8 Download full-text