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Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome

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In October 2009, we reported the first direct isolation of infectious xenotropic murine leukemia virus-related virus (XMRV). In that study, we used a combination of biological amplification and molecular enhancement techniques to detect XMRV in more than 75% of 101 patients with chronic fatigue syndrome (CFS). Since our report, controversy arose after the publication of several studies that failed to detect XMRV infection in their CFS patient populations. In this addenda, we further detail the multiple detection methods we used in order to observe XMRV infection in our CFS cohort. Our results indicate that PCR from DNA of unstimulated peripheral blood mononuclear cells is the least sensitive method for detection of XMRV in subjects' blood. We advocate the use of more than one type of assay in order to determine the frequency of XMRV infection in patient cohorts in future studies of the relevance of XMRV to human disease.
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Virulence 1:5, 386-390; September/October 2010; © 2010 Landes Bioscience
ARTICLE ADDENDUM
386 Virulence Volume 1 Issue 5
Key words: biological amplification,
molecular amplification, XMRV, myalgic
encephalomyelitis/chronic fatigue
syndrome
Submitted: 02/26/10
Revised: 05/20/10
Accepted: 05/24/10
Previously published online:
www.landesbioscience.com/journals/
virulence/article/12486
*Correspondence to: Judy A. Mikovits;
Email: judym@wpinstitute.org
In October 2009, we reported the first
direct isolation of infectious xenotro-
pic murine leukemia virus-related virus
(XMRV). In that study, we used a com-
bination of biological amplification and
molecular enhancement techniques to
detect XMRV in more than 75% of 101
patients with chronic fatigue syndrome
(CFS). Since our report, controversy
arose after the publication of several
studies that failed to detect XMRV infec-
tion in their CFS patient populations. In
this addenda, we further detail the mul-
tiple detection methods we used in order
to observe XMRV infection in our CFS
cohort. Our results indicate that PCR
from DNA of unstimulated peripheral
blood mononuclear cells is the least sen-
sitive method for detection of XMRV in
subjects’ blood. We advocate the use of
more than one type of assay in order to
determine the frequency of XMRV infec-
tion in patient cohorts in future studies
of the relevance of XMRV to human
disease.
Patient selection poses a challenge to
any study of myalgic encephalomyelitis/
chronic fatigue syndrome (ME/CFS). In
our October 2009 paper, samples banked
from 2006 to 2008 were selected for our
study from severely disabled patients who
fulfilled the 1994 CDC Fukuda Criteria
for chronic fatigue syndrome1 as well as
the 2003 Canadian Consensus Criteria
(CCC) for ME/CFS.2 The CCC requires
Detection of an infectious retrovirus, XMRV, in blood cells of patients
with chronic fatigue syndrome
Judy A. Mikovits,1,* Vincent C. Lombardi,1 Max A. Pfost,1 Kathryn S. Hagen1 and Francis W. Ruscetti2
1Whittemore Peterson Institute; Reno, NV USA; 2Laboratory of Experimental Immunology; Cancer and Inflammation Program;
National Cancer Institute-Frederick; Frederick, MD USA
post-exertional malaise, which many cli-
nicians feel is the sine qua non of ME/
CFS. Furthermore the CCC further
requires that patients exhibit post exer-
tional fatigue, unrefreshing sleep, pain
and neurological/cognitive manifesta-
tions, rather than these being optional
symptoms.3 Many clinicians interested in
CFS are switching to the Canadian crite-
ria because they feel it is more descriptive
of the clinical entity being defined. The
Fukuda criteria have the advantage of a
longer period of usage and existence of
many publications that have added modi-
fications. Suffice it to say that the clinician
author of the Science paper elected to use
both criteria, thus bypassing the argument
of which criteria were better. Moreover,
the emphasis in the Science paper was
directed toward the virology, not the clini-
cal description of ME/CFS.
In our October 2009 publication, we
established XMRV infection in the blood
products of our patient population by five
different methods. Of these methods,
single-round PCR on DNA from periph-
eral blood mononuclear cells (PBMCs),
the least sensitive method, required us to
use samples from a subset of chronically ill
patients we had observed to have persis-
tent viremia. In Figure 1A of our Science
paper, we showed that DNA of 7 of 11
patients exhibited the expected gag and
env PCR amplification products from sin-
gle-round PCR with XMRV primers. We
included this figure to demonstrate that
Addendum to: Lombardi VC, Ruscetti FW, Das
Gupta J, Pfost MA, Hagen KS, Peterson DL, et al.
Detection of an infectious retrovirus, XMRV, in
blood cells of patients with chronic fatigue syn-
drome. Science 2009; 326:585–9; PMID: 19815723;
DOI: 10.1126/science.1179052.
www.landesbioscience.com Virulence 387
ARTICLE ADDENDUM ARTICLE ADDENDUM
samples exhibited gag products upon
nested PCR, though PCR with nested env
primers did not result in detectable prod-
ucts from these samples (Table 1).
Samples that are negative for XMRV
by one of our PCR assays are sometimes
positive by other assays. For example, in
Figure 1A of the Science paper, patient
1118 was negative by single round PCR
on DNA from unstimulated PBMCs, but
positive in other assays (Science Figs. 2A
and D, 4A and S5). Of the 34 patients
whose PBMCs were negative for XMRV
by DNA or cDNA PCR, 17 were posi-
tive for infectious virus when co-cultured
with the LNCaP indicator cell line, as
XMRV gag and env PCR products were
detected in the cell line following their
infection with XMRV from the patient
PBMCs (Tab le 2 ). Both gag and env prod-
ucts obtained from either single-round or
nested PCR were sequenced and shown to
be 99% identical to XMRV VP62.
Subsequent to our October 2009
publication, two papers from the United
Kingdom4,5 and a paper from the
Netherlands6 have appeared in which
the authors report the lack of detection
of XMRV PCR products from DNA of
unstimulated PBMCs, using patient pop-
ulations selected by only the Fukuda crite-
ria or the Oxford criteria rather than both
Fukuda and CCC criteria. We regret that
these authors did not request positive con-
trol samples of our patients who exhibit
XMRV PCR products even when assayed
by the least sensitive detection method,
namely PCR of DNA from unstimulated
PBMCs. Given that only 7% of our 101
patients’ PBMCs exhibit products upon
DNA PCR (Table 3 and 4), and that a
number of patients were included in the
UK studies who do not fulfill the CCC
criteria, very few, if any, of the samples
would be expected to be positive by DNA
PCR. We also note that both studies fol-
lowed different methods than ours for
blood collection, DNA quantities and
isolation and PCR, possible sources of
the disparate results. The XMRV detec-
tion results of the 101 patients are listed
in Table 4.
The negative reports of PCR tests for
XMRV has raised questions whether our
findings could be due to contamination of
our PCR experiments by mouse genomic
XMRV-gag specific PCR products and no
env specific PCR products following single
round DNA PCR of DNA of unstimu-
lated PBMCs. In contrast, when cDNA
was prepared from PBMCs, 67% of the
nested PCR, which inevitably raises ques-
tions of contamination, is not essential
to detect XMRV in highly viremic ME/
CFS patients. The remaining 90 samples
described in the paper exhibited very few
Tab le 1. XMRV detection using cDNA from 22 unstimulated PBMCs
gag gene env gene
Sample 1st 2nd Sample 1st 2nd
1 - - Normal 1 - - Normal
2 - + 1104 2 - - 110 4
3 + + 1110 3 - - 1110
4 - - 1113 4 - - 1113
5 - - 1114 5 - - 1114
6 - + 1115 6 - + 1115
7 - - 1117 7 - - 1117
8 - + 1125 8 - - 112 5
9 - + 1130 9 - - 113 0
10 + + 1135 10 - - 1135
11 - - 11 42 11 - - 1142
12 + + 1150 12 - - 115 0
13 - - 1155 13 - - 115 5
14 - + 1161 14 - - 1161
15 - + 1165 15 - - 116 5
16 - + 1166 16 - - 116 6
17 + + 1168 17 - - 116 8
18 - + 1169 18 - - 116 9
19 - + 1177 19 - - 117 7
20 - + 1178 20 - - 1178
21 - - 1182 21 - - 118 2
22 - - 1199 22 - - 119 9
Tab le 2 . Co-culture with LNCaP of PBMCs from 12 patients PCR negative for env
gag gene env gene
Sample 1st 2nd Sample 1st 2nd Typ e
1 - - Normal 1 - - Normal cDNA
2 - + 116 9 2 + + 1169 cDNA
3 + + 1221 3 + + 1221 cDNA
4 - + 1150 4 + + 1150 cDNA
5 - + 119 9 5 - + 119 9 cDNA
6 + + 1220 6 + + 1220 cDNA
7 - - LNCaP 7 - - LNCaP cDNA
8 - + 118 6 8 - + 118 6 cDNA
9 - + 1132 9 - + 1132 DNA
10 - + 1111 10 - - 1111 DNA
11 - + 1189 11 + + 118 9 DNA
12 - + 1172 12 + + 117 2 DNA
13 - + 1173 13 - + 117 3 DNA
14 - + 1103 14 + + 1103 DNA
388 Virulence Volume 1 Issue 5
Tab le 4. XMRV detec tion results of 101 patients
Patient ID cDNA nested PCR DNA nested PCR LNCaP co-culture with
PMCs
Antibody in
plasma
LNCaP culture with
plasma
110 3 + + + + +
110 4 + + + + +
110 5 + - + + +
110 6 + + + + +
1107 + - - NT*NT
110 8 + - - - -
110 9 + - NT NT NT
1110 + - + + +
1111 + + + - +
1112 + - NT NT NT
1113 + - + NT NT
1114 + - NT NT +
1115 + - + + +
1116 - - NT NT +
1117 - - NT NT NT
1118 + - + + +
1119 + - NT NT NT
1120 - - NT NT NT
1121 + - NT NT NT
1124 + - - - -
1125 + - + + +
1126 + - NT NT NT
1127 + - NT NT NT
1128 + - NT NT NT
1129 + - NT -NT
1130 + - NT NT NT
1131 + - NT NT NT
1132 + + + NT NT
1133 + + NT NT NT
113 4 - - NT NT NT
1135 + + NT NT NT
1136 + + - + +
1137 + + - + +
1265 + - + + +
1138 + - NT NT NT
1335 + - NT + +
1139 - - - - -
*NT, not tested. Note not all assays were run on all samples and/or patients.
Tab le 3. Summary of multiple viral assays from a group of 57 patients
Unstimulated PBMC Stimulated PBMC Co-Cultured LNCaP Serology Unstimulated PBMC
Nested gag Nested gag Nested gag Env antibody Single round gag
cDNA DNA cDNA -cDNA -Plasma DNA
31/57 44/205*41/57 -51/57 -47/57 4/57
54% 21% 72% -89% -82% 7%
*multiple DNA samples taken from some of the 57 patients on different dates.
www.landesbioscience.com Virulence 389
Tab le 4. XMRV detec tion results of 101 patients
114 0 + - NT - +
1141 + - + + +
1142 - - NT NT +
120 6 + - NT - +
114 4 + - NT NT NT
1145 - - NT NT NT
114 8 - - NT NT NT
1149 + - NT NT NT
1150 + + + + +
1151 + - NT NT NT
123 0 + - + NT NT
1237 + - + NT NT
115 4 - - NT NT NT
1155 - - NT NT NT
1156 - - NT NT +
1157 + + NT NT NT
1158 + - - + +
1159 + - NT NT NT
1231 + - + NT NT
1161 + - - + +
1220 + - + NT NT
1221 + - + NT +
116 4 - - NT NT NT
116 5 + - + + +
116 6 + - - + +
1167 - - NT NT NT
116 8 + - NT NT NT
116 9 + - + + +
1170 - - NT NT NT
1235 - - + NT NT
1281 + - + + +
1172 + + + + +
1282 + - - - +
1173 + + + + +
1174 + - NT NT NT
1175 - - NT NT NT
1176 - - NT NT NT
1177 + - + + +
1178 + - + + +
1179 + - NT - +
118 0 - - NT + +
1181 + - NT NT NT
118 2 - - NT + +
118 3 + - - - +
123 6 + - + NT NT
1224 + - NT NT +
118 6 + + + + +
*NT, not tested. Note not all assays were run on all samples and/or patients.
390 Virulence Volume 1 Issue 5
unless PCR primers are designed with this
possibility in mind.
We have not claimed in our October
2009 publication or in other venues that
XMRV is the cause of CFS, only that its
detection in the majority of our ME/CFS
patient cohort allows us to form a testable
hypothesis as to an infectious basis for this
devastating disease. Future work should
establish what role XMRV may play in
development of prostate cancer, ME/CFS
and other diseases.
References
1. Fukuda K , Straus S, Hickie I, Sharpe MC, Dobbins
JG, Komaroff A. The chronic fatigue syndrome: A
comprehensive approach to its definition and study.
Ann Intern Med 1994; 121:953-9.
2. Carrut hers B, Jain A, DeMeirlier K, Peterson DL ,
Klimas NG, Lerner AM, et a l. Myalgic encephalo-
myelitis/chronic fatigue syndrome: Clinical work ing
case definition. diagnostic and t reatment protocols. J
Chronic Fatigue Syndrome 2003; 11:1-12.
3. Jason L, Torres-Harding S, Jurgens A, Helgerson
J. Comparing the Fukuda et al. criteria and the
Canadian def inition for chronic fatigue sy ndrome. J
Chronic Fatigue S 2004; 12:37-52.
4. Erlwein O, Kaye S, McClure MO, Weber J, Wills G,
Collier D, et al. Failure to detect the novel retrovirus
XMRV in chronic fatigue syndrome. PLoS One
5: 8519.
5. Groom HC, Boucherit VC, Makinson K, Randal
E, Baptista S, Hagan S, et al. Absence of xenotropic
murine leu kaemia virus-related virus in UK patients
with chronic fatigue syndrome. Retrov irology 7.
6. van Kuppeveld FJ, de Jong AS, La nke KH, Verhaegh
GW, Melchers WJ, Swanink CM ,et al. Prevalence of
xenotropic murine leukaemia virus-related virus in
patients with chronic fatigue syndrome in t he
Netherlands: retrospective ana lysis of samples from
an established cohort. BMJ 340:1018.
7. Ratner L, Philpott T, Towbridge DB. Nucleotide
sequence a nalysis of isolates of Human
T-lymphotropic virus type 1 of diverse georgraphical
regions. AIDS Rs Hum Retroviruses 1991; 7:923-41.
8. Verdonck K, Gonzalez E, Van Dooren S, Vandamme
AM, Vanh am G, Gotuzz o E. Human T-lymphotro pic
viru s 1: Recent knowled ge about an ancient i nfection.
Lancet Infect Dis 2007; 7:266-81.
9. Yan Y, Liu Q, Kozak C A. Six host range variants of
the xenotropic/polytropic gammaretroviruses def ine
determinants for entry in the XPR1 cell surface
receptor. Retrovirology 2009; 6: 87.
In our experience from performing the
multiple methods on the same 57 blood
samples, the most sensitive blood-based
assays for detection of XMRV in decreas-
ing order (Tabl e 3) are: (1) perform-
ing nested PCR for gag sequences from
LNCaP cells that have been co-cultured
with subject’s plasma or activated PBMCs,
(2) the presence of antibodies to XMRV
Env in subject’s plasma, (3) presence of
gag products by nested PCR on stimu-
lated PBMCs or detection of viral pro-
teins expressed by activated PBMCs with
appropriate antisera, (4) nested RT-PCR
of plasma nucleic acid or PCR from cDNA
from unactivated PBMCs and (5) PCR of
DNA from unactivated PBMC prepared
from subject’s blood.
Despite association with both prostate
cancer and CFS, many questions remain
regarding the prevalence of XMRV in
the human population, the incidence
of XMRV in disease, and the extent of
genetic variation between XMRV isolates.
The genetic variation between XMRV
isolates currently identified is only 0.03%,
despite the fact that the viral sequences
were obtained from isolates from two
vastly different diseases in patients from
geographically distinct areas. This varia-
tion is smaller than the variation observed
between HTLV-1 isolates.7 As in the case
with HTLV, the lack of diversity implies
that XMRV recently descended from a
common ancestor.8 The high degree of
similarity to xenotropic murine leukemia
virus suggests that a cross-species trans-
mission event was likely involved in the
evolution of XMRV.9 Further examina-
tion of XMRV from human subjects may
reveal more extensive sequence variation,
which also may confound its detection
DNA, which contain gag and env sequences
highly similar to XMRV. Positive PCR
results for XMRV were obtained inde-
pendently in multiple laboratories led by
co-authors of the Science paper. In the sum-
mer of 2006, prior to work on XMRV at
the Reno Whittemore-Peterson Institute
(WPI), 30 mL of heparinized peripheral
blood were obtained from patients resid-
ing in the US, Canada and Europe com-
ing to be treated at the well-known Sierra
Internal Medicine practice, located in
Incline Village, NV. Once collected, 48 of
these blood samples were shipped directly
to NCI where cDNA was prepared for
planned microarray experiments. After the
WPI observed an XMRV PCR product
from a patient sample in 2009, the NCI
began testing these stored samples by PCR.
cDNA from 42 of the 48 samples sent to
the NCI lab in February 2007 tested posi-
tive for XMRV gag by nested PCR. Neither
the WPI nor NCI labs where PCR was per-
formed had ever worked with mouse tissues
or had been exposed to XMRV from other
sources. The env sequences amplified from
LNCaP cells infected by patient PBMCs
exhibit less similarity to mouse genomic
DNA than to XMRV VP62, further indi-
cating the presence of XMRV infection
rather than mouse genomic DNA contami-
nation. After we developed a sensitive cell
culture assay for detection of XMRV, we
assayed our cell lines and patient material
with a highly sensitive assay (developed and
kindly provided by Bill Switzer, CDC) to
detect the presence of mouse tissue con-
tamination by the identification of murine
mitochrondial cytochrome oxidase by real
time PCR. All of the cell lines and 101
patient materials tested negative for mouse
contamination.
Tab le 4. XMRV detec tion results of 101 patients
1187 - - NT + +
118 8 + - + + +
118 9 + + + + +
119 0 + - + + +
1191 + - + + +
1192 + + NT + +
1193 + + NT + +
119 4 + - NT - +
123 8 + - + + +
*NT, not tested. Note not all assays were run on all samples and/or patients.
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Recent years have brought growing recognition of the need for clinical criteria for myalgic encephalomyelitis (ME), which is also called chronic fatigue syndrome (CFS). An Expert Subcommittee of Health Canada established the Terms of Reference, and selected an Ex- pert Medical Consensus Panel representing treating physicians, teaching faculty and researchers. A Consensus Workshop was held on March 30 to April 1, 2001 to culminate the review process and establish consensus for a clinical working case definition, diagnostic protocols and treatment protocols. We present a systematic clinical working case definition that