Murine Gammaretrovirus Group G3 Was Not Found in
Swedish Patients with Myalgic Encephalomyelitis/
Chronic Fatigue Syndrome and Fibromyalgia
Amal Elfaitouri1, Xingwu Shao1{, Johan Mattsson Ulfstedt1, Shaman Muradrasoli1, Agnes Bölin Wiener1,
Sultan Golbob1, Christina Öhrmalm1, Michael Matousek2, Olof Zachrisson2, Carl-Gerhard Gottfries2,
Jonas Blomberg1*
1 Section of Clinical Virology, Department of Medical Sciences, University of Uppsala, Uppsala, Sweden, 2 Gottfries Clinic AB, Institute of Neuroscience and Physiology,
University of Gothenburg, Gothenburg, Sweden
Abstract
Background: The recent report of gammaretroviruses of probable murine origin in humans, called xenotropic murine
retrovirus related virus (XMRV) and human murine leukemia virus related virus (HMRV), necessitated a bioinformatic search
for this virus in genomes of the mouse and other vertebrates, and by PCR in humans.
Results: Three major groups of murine endogenous gammaretroviruses were identified. The third group encompassed both
exogenous and endogenous Murine Leukemia Viruses (MLVs), and most XMRV/HMRV sequences reported from patients
suffering from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Two sensitive real-time PCRs for this group
were developed. The predicted and observed amplification range for these and three published XMRV/HMRV PCRs
demonstrated conspicuous differences between some of them, partly explainable by a recombinatorial origin of XMRV.
Three reverse transcription real-time PCRs (RTQPCRs), directed against conserved and not overlapping stretches of env, gag
and integrase (INT) sequences of XMRV/HMRV were used on human samples. White blood cells from 78 patients suffering
from ME/CFS, of which 30 patients also fulfilled the diagnostic criteria for fibromyalgia (ME/CFS/FM) and in 7 patients with
fibromyalgia (FM) only, all from the Gothenburg area of Sweden. As controls we analyzed 168 sera from Uppsala blood
donors. We controlled for presence and amplifiability of nucleic acid and for mouse DNA contamination. To score as
positive, a sample had to react with several of the XMRV/HMRV PCRs. None of the samples gave PCR reactions which
fulfilled the positivity criteria.
Conclusions: XMRV/HMRV like proviruses occur in the third murine gammaretrovirus group, characterized here. PCRs
developed by us, and others, approximately cover this group, except for the INT RTQPCR, which is rather strictly XMRV
specific. Using such PCRs, XMRV/HMRV could not be detected in PBMC and plasma samples from Swedish patients suffering
from ME/CFS/FM, and in sera from Swedish blood donors.
Citation: Elfaitouri A, Shao X, Mattsson Ulfstedt J, Muradrasoli S, Bölin Wiener A, et al. (2011) Murine Gammaretrovirus Group G3 Was Not Found in Swedish
Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Fibromyalgia. PLoS ONE 6(10): e24602. doi:10.1371/journal.pone.0024602
Editor: Yuntao Wu, George Mason University, United States of America
Received May 11, 2011; Accepted August 15, 2011; Published October 12, 2011
Copyright: ß 2011 Elfaitouri 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: This work was supported by ME Research UK (Charity number SC 036942), and the Irish ME Trust and stiftelsen Lars Hiertas minne (FO2010-0672). 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: Jonas.Blomberg@medsci.uu.se
{ Deceased.
than just XMRV [3]. It is known that endogenous retroviral
sequences (ERVs) highly related to XMRV and HMRV occur in
the mouse genome. In fact, XMRV was recently reported to be a
recombinant between two MLV-related murine ERVs [4].
However, beyond that, a more exact mapping between the
MLV-related sequences found in humans, and the murine ERVs,
and indeed the clustering of intact murine gammaretroviral
proviruses has not been performed. We will in the following refer
to the gammaretroviruses related to murine ERVs which have
been reported to occur in humans as ‘‘XMRV/HMRV’’. It is also
unknown if XMRV/HMRV is confined to the mouse genome.
Thus, the origin and spread of XMRV/HMRV can be studied
bioinformatically in the genomes of the mouse and other
Introduction
A gammaretrovirus related to the Mouse Leukemia Viruses
(MLVs), was 2006 found in a few percent of patients suffering from
prostate cancer [1]. It was initially named XMRV, ‘‘Xenotropic
Murine retrovirus Related Virus’’. In 2009 XMRV was also found
in patients suffering from ME/CFS [2]. Although the width of the
term ‘‘XMRV’’ can be understood in a rather broad way, it is
often used in a more restricted sense, as a specific xenotropic
gammaretrovirus that was found in humans with these diseases. In
2010, the term XMRV was complemented with ‘‘HMRV’’
(Human retrovirus related to Murine RetroVirus), because
gammaretroviral sequences found in ME/CFS were more diverse
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300 proviruses contained 35 which had no such mutations, being
‘‘intact’’ by bioinformatic criteria. The 35 had a less than 0.5%
LTR divergence. They are marked with green in the Pol trees
shown in Information S1. Thus, the 35 proviruses had hallmarks
of being infectious, and also belonged to the most recently
integrated murine ERVs.
The phylogenetic analysis of the 300 proviruses together with
related high-scoring gammaretroviruslike proviruses of other
vertebrates is presented in Figure 1 (more detailed in Information
S1). Briefly, three major groups of high scoring murine
gammaretroviral proviruses resulted. The three groups were
evident in both gag and pol nucleotide based trees. The gag and
pol nucleotide based groups contained the same or almost the same
members. Group G1 (Gamma 1) contained 188 members, with an
average within-group identity of 94%. Ten of them had an ORF
in gag, pro, pol and env. Members encompassed the MmERV [22]
(interpreted by RetroTector from GenBank Id AC005743)
sequence. Mus dunni ERV (MdERV, AF053745) was highly
related. The most similar non-mouse proviruses were the rat
Chr17 5186121 and Chr7 31839324 ERVs, and more distantly,
Gibbon ape Leukemia Virus (GaLV, PCGGPE) and Koala
RetroVirus (KoRV, AF151794) sequences. Group G2 contained
59 members, with an average within-group identity of 85%. Three
of them had an ORF in gag, pro, pol and env). It contained the GLN
murine ERVs of Ribet et al [23]. Outside of the mouse genome, a
group of PERV located at chr9 151463024, chr10 71670155,
chr12 29233668 and chr4 47233287, respectively, were highly
(78–87% identical) related. More distantly related (73–78%
identical) were, rat endogenous proviruses on chr9 2586497 and
chr7 74285691. Group G3 contained 53 members, with an
average within-group identity of 93%. Twentytwo had an ORF in
gag, pro, pol and env. It encompassed the eco-, xeno-, poly- and
modified polytropic endogenous MLVs [24,25,26]. The ecotropic
exogenous MLVs had only one endogenous G3 counterpart, on
chromosome 8 (see Figures 1 and Information S1).
Exogenous ecotropic MLVs were 86–94% identical to the
consensus of the endogenous G3 members. They can therefore
also be considered to be G3 viruses. The amphotropic MLVs were
not represented among the endogenous sequences, but clustered at
the base of G3. The Hortulanus endogenous murine provirus
(HEMV) [27] was ancestral to the G3 group (not shown here) [13].
The properties of the G1–G3 groups can be further studied in
Information S3 and Table S1. The degree of coherence of the
groups, consensus sequences and PBS usage are shown in
supporting document S3. The relationships between the MLLVs
will be further examined in a forthcoming paper [13]. The three
major groups were discernible in neighbor joining (NJ) trees
resulting from alignment of gag, pol and env nucleotide sequences,
and, with 99–100% bootstrap support, in NJ and minimum
evolution (ME) trees as well as in maximum likelihood (ML) trees,
resulting from Gag, Pol and Env protein alignments, using the
MEGA 5 phylogeny package and the ClustalW and MUSCLE
alignment programs. They represent three different, evolutionary
recent, bursts of gammaretroviral proliferation in the mouse and
its immediate progenitors. The third group, highly similar to the
retroviruses reported from the human diseases prostate cancer and
ME/CFS, contains the highest proportion of bioinformatically
intact proviruses. It may thus have the greatest zoonotic potential.
The data set of 300 high scoring murine gammaretroviruslike
proviruses, plus reference MLV sequences from GenBank, and
related sequences from other species in the ‘‘RetroBank’’
collection [20], was the basis of the bioinformatic prediction of
the detection range of the PCRs employed in this work.
vertebrates. In principle, the reports of XMRV/HMRV in
humans with and without disease could have far-reaching
implications, for the personal life of the patients, for the
development of diagnostic methods, for transfusion safety, and
for the understanding of other human diseases with a possible
retroviral etiology. Reports which verify [3] and do not verify [5,6]
the original ME/CFS report have come. The conflicting results
may be due to methodological differences (some were demonstrated here), an uneven geographic distribution of XMRV/
HMRVs, or different types of laboratory contamination
[4,7,8,9,10,11,12].
MLVs are gammaretroviruses which may be both exogenous
(infects between individuals of a similar generation, i.e. horizontally), or endogenous (proviruses integrated into the germ line of
mice and were thereby transmitted to the next generation, i.e.
vertically). However, a wider group of MLV-like gammaretroviruses (‘‘MLLVs’’) [13] have been, and are, spreading among
vertebrates. They have repeatedly infected nonmurine vertebrates
in the not so distant past. Gibbon apes [14,15] and koalas [16]
have been ‘‘invaded’’ by MLLVs. A similar transspecies
gammaretroviral infection of uncertain origin occurred recently
in birds [17]. MLLVs include the gammaretroviruses of
mediterranean and middle eastern cats [18], and of pigs, although
the murine origin of the virus in those species is less certain. In the
infected animals, MLLVs are associated with significant disease
like encephalitis, malignancy (leukemia and lymphoma), wasting,
and immunosuppression. Is the human species now also ‘‘invaded’’
by an MLLV, i.e. XMRV/HMRV?
ME/CFS can be diagnosed according to internationally
accepted criteria, see e.g. [19]. It seems to be a rather common
disease, maybe amounting to 0.4% of the population [19]. Finding
the cause, new diagnostic techniques and, hopefully, a cure, for
this often debilitating disease is a high medical priority. ME/CFS
borders to the diseases fibromyalgia (FM) and irritable bowel
syndrome (IBS). Evidently, there is a great need for confirmation
of the reports on XMRV/HMRV in ME/CFS, and in other
populations. In view of the recently reported diversity of retroviral
sequences in ME/CFS, it is important to establish the detection
range of XMRV/HMRV detection methods.
In the present study, we first address murine gammaretroviral
diversity. We describe three groups among them. We trace the
recombinatorial origin of XMRV using murine endogenous
retroviral sequences. From this we predict the ability of our own
and of some other commonly used XMRV/HMRV PCRs to
detect portions of this murine gammaretroviral spectrum. We
report the development of two sensitive PCRs which are broadly
targeted to detect murine retroviruses s belonging to group G3.
We further apply those two and some previously published
XMRV/HMRV detection PCRs to PBMCs from ME/CFS
patients and sera from blood donors. We also address the possible
occurrence of contamination, either from the PCRs themselves,
from synthetic target DNA, or from murine DNA.
Results
Gammaretroviruslike proviruses of the genomes of the
mouse and some other vertebrates, as represented in the
‘‘RetroBank’’ collection
Of 7656 retroviral sequences detected in the mm8 assembly,
1461 were gammaretroviruslike [20]. Some of the latter (300) were
scored higher than 2000 by RetroTector (ReTe) [21]. This meant
that they were structurally intact or almost intact. They were all
complete proviruses in which very few stop or shift (indel)
mutations likely incapacitating the provirus were detected. The
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Figure 1. gag sequences of 300 high scoring mouse gammaretroviral sequences were aligned together with reference sequences.
The tree was rooted with a rabbit sequence. Sequences in red are from ME patients, published in the paper of Lo et al [3]. Two short blood donor
(‘‘BD’’) sequences from the Lo et al study came out at the beginning of group G2, in most other trees in group G3. ‘‘HMRV’’ corresponds to group G3
in the tree. A higher resolution version is shown in Information S1). (genomic ERV sequences taken from the prototype of RetroBank were named as
oryCun = rabbit, cavPor = guinea pig, felCat = Cat, panTro = Chimpanzee or rheMac = Rhesus macaque. Mouse sequences from the mm8 assembly are
just shown with their chromosomal location). MLV sequences of known tropism, from GenBank, were also added. The separation of xeno-, modified
polytropic and polytropic G3 sequences was not clear in this tree. It was more clear in other trees (Information S1).
doi:10.1371/journal.pone.0024602.g001
The unique recombinatorial origin of 22Rv1/XMRV
studied using murine ERVs
PreXMRV-1 and -2 likely derived from the nude mice which
were used to propagate the 22Rv1 cells. These mice have their
own variant set of ERVs. However, the four sequences most
similar to each of the four were selected, giving 14 XMRV/
22Rv1-related sequences, all falling within group G3. XMRV and
22Rv1 were 99.9% identical, and gave the same results in all
comparisons with the 14 related sequences. A similarity plot of
XMRV/22Rv1 versus PreXMRV-1, PreXMRV-2 and the group
G3 consensus sequence (Figure 2a and b) showed that indeed
PreXMRV-1 was 99% identical to XMRV/22Rv1 over the
middle and the LTRs, while PreXMRV-2 was 99% identical to
XMRV/22Rv1 over 3600 nucleotides covering gag, pro and a
part of pol, and two shorter regions just before the LTR. No other
It was recently shown that the XMRV that was found in
samples from humans suffering from prostate cancer and ME/
CFS most likely is a recombinant between two murine
gammaretroviral ERVs, PreXMRV-1 and PreXMRV-2 which
arose during passage of the human prostate cancer cell line 22Rv1
in nude mice [4]. We used the mm8 sequences in RetroBank to
verify and extend this conclusion. We first searched for the most
related sequences to PreXMRV-1, PreXMRV-2, 22Rv1 and
XMRV in RetroBank using BlastN, and in the murine genome
and nonredundant sequence data sets in GenBank, using
MegaBlast. There were no exact counterparts to them.
Figure 2. Relation between the XMRV/HMRV PCRs considered in this paper, and the recombinant origin of XMRV/22Rv1. Similarity
plots from a Clustal alignment with 22RV1 (Fig. 2A) and XMRV (Fig. 2B) as references, and with PreXMRV-1, PreXMRV-2 and the G3 consensus (from
this paper) as queries, were made with Simplot v1.3 (1998, kindly provided by dr S. Ray, dpt of Infectious Diseases, Johns Hopkins University, US) with
a window size of 100 and a step size of 20 nucleotides, inclusion of gaps and Kimura transition/transversion scoring. The 22Rv1 virus was 99.9%
identical to XMRV over the entire alignment, and yielded an identical similarity plot with the probable ancestors and the G3 consensus. The mosaic
nature of XMRV, and its consequences for the detection range of the PCRs considered in this paper, as well as the exact match between 22Rv1 and
XMRV, is evident. It is highly unlikely that the same recombination could have occurred by chance independently, outside of the 22Rv1 cell line.
Additional material is found in Information S2.
doi:10.1371/journal.pone.0024602.g002
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hybrid sequences intermediate between HERV-T and XMRV
were made.
The results (Figure 4) showed that the murine chromosome 4
(belonging to group G2), chromosome 10 (group G1) and 13
(group G2) and HERV-T proviral sequences were only detected at
100–10000 times higher concentrations than the XMRV
sequence, respectively. Thus, the predictions shown in figure 3
were approximately corroborated. The broadly targeted gag
RTQPCR presented in this paper will probably miss murine
gammaretroviruses outside of group G3.
To make a similar study of the targets of the env RTQPCR, a
panel of artificial hybrid synthetic env sequences, with a gradual
transition between chr13 6814088 (belonging to group G2) and
XMRV VP62, was synthesized (Figure 5). The five synthetic
targets were tested with the env RTQPCR. The results (Figure 5)
showed that the provirus at chr13 68140880, only could be
inefficiently amplified. It was also predicted to be amplifiable
neither by the env RTQPCR, nor by any other of the evaluated
PCRs (Figure 3).
sequences with such a high degree of similarity to XMRV/22Rv1
were identified in RetroBank or GenBank.
The regional distribution of similarity between PreXMRV-1,
PreXMRV-2 and their most similar proviruses from the databases,
and the uniqueness of 22Rv1/XMRV among xenotransplantation
derived xenotropic viruses is further detailed in Information S2.
The region around 5000 (target for the INT RTQPCR mentioned
below) seems to be a major dissimilarity region within group G3
genomes (cf. Figure 2). The mosaic origins of 22Rv1/XMRV thus
have a bearing on the detection range of the PCRs whose targets
are distributed over much of the 22Rv1/XMRV genome (see
below and Figure 2).
Design and evaluation of the gag and env RTQPCRs
The design considerations, and the basic methodological
evaluation, for the gag and env RTQPCRs are detailed in the
Information S4.
Bioinformatic prediction of detection range for XMRV/
HMRV PCRs
Evaluation of sensitivity and specificity
None of the PCRs amplified from human DNA. The env and gag
RTQPCRs had a sensitivity of 1–10 copies of XMRV VP62
plasmid and synthetic target DNA in dilution experiments. The
INT RTQPCR did not amplify from two different (Balb/C and
C3H) mouse DNA samples. The two xenotropic endogenous
sequences at chr1 173317855 and chr1 172778230 of C57Black/6J,
predicted to be amplifiable, may not be present in these mice. The
env and gag RTQPCRs, as well as the Lo/Alter primers, amplified
from mouse DNA. In several tenfold dilution experiments (not
shown), the env and gag RTQPCRs, and the nested Lo/Alter
primers, could detect 0.1 - 0.01 copies of mouse DNA, whereas the
non-nested outer Lo/Alter primers could detect around 1 copy of
mouse DNA. The nucleic acid extracts of samples from patients and
blood donors had a variable nucleic acid content, as estimated by
the His3.3 RTQPCR: Blood donor sera contained 0.17–116, ME/
CFS/FM PBMC samples 0.06–1017, and ME/CFS/FM plasma
0.53–15.9 ng amplifiable nucleic acid/PCR reaction. Thus, a
positive outcome of XMRV/HMRV PCRs could not be expected
from all samples due to a low RNA/DNA concentration (Figure 6).
An approximate prediction of PCR detection range among the
aligned sequences was made using a computer program based on
the NucZip algorithm [28]. Its output is a detection ability score,
shown as horizontal bars for each provirus in an alignment of the
300 intact or nearly intact proviruses in the mm8 assembly. As
seen in figure 3, results with both the outer primers of the nested
Lo/Alter PCR, and the primers and probe of the gag RT QPCR
presented here, indicate that it is only the third group (which
contains the ‘‘traditional’’ MLV-like proviruses) that can be
detected with these PCRs. The predicted detection range of the
INT RTQPCR, which uses two different reverse primers, is much
more narrow (Figure 3d). The nonendogenous variants of XMRV
isolated from humans and human cells (VP62, VP35 and 22Rv1)
were indicated to be detectable. However, two highly XMRVrelated proviruses on chromosome 1 of C57Black/6J mice (the
source of the mm8 assembly) were also indicated to be detectable
(chr1 173317855; XMV43 and chr1 172778230; XMV41). The
XMV subgroup assignments are from Jern et al [26]. These are
the same sequences which were noted to be highly related to
PreXMRV-1 in the Information S2. Thus, the C57Black/6J
mouse genome contains a few sequences predicted to be detectable
with the INT RTQPCR. The reverse transcriptase - directed
nested primers of Switzer et al [5] also seem to cover most of group
G3. Finally, the predicted detection range of the env RTQPCR,
with its variation tolerant MegaBeacon probe, covered most of the
group G3 murine gammaretroviruses, except for the ecotropic
ones (Figure 3 e). All these PCRs, except for the one of Switzer
et al [5], were tested in this paper.
Results with the three RTQPCRs and human samples
gag RTQPCR. This PCR yielded no positive samples out of
85 ME/CFS/FM samples from Gothenburg. However, two
samples (one from PBMC and one from plasma) reacted weakly
initially, but sequencing revealed that this weak signal was due to
contamination from the synthetic positive control with an abridged
XMRV sequence shown in figure 4. This artificial sequence is
highly unlikely to be of natural origin. None of 168 Blood donor
samples from Uppsala were positive. However, 11 were initially
weakly reactive (3–16 copies per PCR reaction, Ct 38.1–41.3).
Sequencing revealed the same artificial sequence as mentioned in
the previous section.
All PCR results except for the His3.3 RTQPCR results are
summarized in Table 1.
env RTQPCR. None of the 85 ME/CFS/FM patients from
Gothenburg and 168 Blood donor serum samples from Uppsala
were positive in the env RTQPCR, neither were they weakly
reactive.
INT RTQPCR. Similarly, none of 168 blood donor serum
samples from Uppsala, PBMC and plasma samples from 85 ME/
CFS/FM patients in Gothenburg were positive in the INT
RTQPCR. However, one out of 168 blood donor serum samples
from Uppsala was weakly initially reactive with Ct = 41.92. One
Test of detection range for the gag and env QPCRs using
synthetic targets
The gammaretroviral sequence closest related to XMRV/
HMRV in the human genome is HERV-T [29,30]. We wanted to
test how well separated the mouse gammaretroviral group G3
sequences were from HERV-T and from mouse gammaretroviral
groups G1 and G2, by systematically letting primer and probe
target sequences vary from murine to human sequence. Thus,
gammaretroviral gag sequences which included XMRV and
murine gammaretroviruses from mouse chromosome 4, 10 and
13, as well as HERV-T from human chromosome 11, were
aligned. In order to test the detection range of the gag RTQPCR
synthetic target sequences from aligned portions of these sequences
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MERV G3: In Mouse Genome but Not in Patients
Figure 3. Estimated PCR detection range in an alignment of 300 high-scoring gammaretroviruslike proviruses found by
RetroTector in the mm8 assembly. Predictions were mapped onto NJ trees of alignments of gag, pol, integrase and env nucleotide sequences. A
PCR detection score (shown as horizontal bars) was calculated by multiplying the fit of primers and probe (if present) to the target sequences for each
provirus in the alignment. The degree of fit (match) was estimated using a modified NucZip algorithm [28]. A more complete treatment of this
subject will be published separately (Danielsson et al, in preparation). The trees and alignments are further presented, and shown in higher resolution
together with trees made with several algorithms, with bootstrap figures, in Information S1. A. A gag nt alignment assessed with outer primers of the
nested Lo/Alter PCR [3] (HMRV sequences from that paper are shown in red), and B. the same tree with the prediction for the primers and probe of
the gag RTQPCR presented in this paper. C. A pol tree, with the prediction for the outer primer pair of the nested RT-based PCR of Switzer et al [5] is
shown. D. A tree based on the 39 (mainly integrase) portion of pol. Predictions based on the Singh RTQPCR, with its two reverse primers [35], are
shown. XMRV sequences are shown in red. E. An env tree. The prediction of the detection range of the env RTQPCR presented in this paper is shown.
A higher resolution picture is shown in Information S1.
doi:10.1371/journal.pone.0024602.g003
plasma sample from the 85 ME/CFS/FM patients from
Gothenburg was also weakly initially reactive, with Ct = 41.41.
None of these two was repeatable.
ME/CFS/FM PBMC samples (median 197 ng/PCR reaction)
contained most amplifiable nucleic acid, followed by the 168 blood
donor sera (median 12.3 ng/PCR reaction) and the 49 ME/CFS/
FM plasma samples (median 2.5 ng/PCR reaction).
In order to avoid false negative results, a desirable amount of
DNA per PCR reaction is 100 ng (DNA from approximately
15000 cells). This limit is plotted in figure 6, using an approximate
conversion from amplifiable nucleic acid found by the His3.3
Assessment of amplifiable nucleic acid in the human
samples
As seen in figure 6, the histone 3.3 RTQPCR revealed a
variable amount of amplifiable nucleic acid in the samples. The 85
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Figure 4. Alignment of endogenous group G1 (chr10) and G2 (chr4 and 13) MERV sequences, HERV-T and artificial hybrid HERV-T/
XMRV target sequences used to test the detection range of the gag RTQPCR. The HERV-T sequence was gradually changed (red
nucleotides) to become more XMRV-like. The detection limit was determined by running PCR with tenfold dilutions giving 1–100 000 molecules per
PCR reaction. The lowest positive concentration, in molecules per PCR reaction of the respective constructs, is written to the right of the alignment.
The number of mismatches (underlined) between forward primer, probe and reverse primer, respectively, is shown at the far right.
doi:10.1371/journal.pone.0024602.g004
RTQPCR. In the PBMC samples, around 40% were estimated to
contain more than 500 ng DNA per PCR reaction. The weakly
reactive RTQPCR results in the three sets, with the exception of
the gag RTQPCRs which were shown to be due to contamination
(Table 1) are shown as red arrows. The sample positive for mouse
DNA contamination is shown with a blue arrow.
the weakly reactive samples from the screening RTQPCRs.
However, the eleven weakly gag reactive samples were proven to
arise from contamination with a synthetic target DNA. The two
weakly INT RTQPCR reactive samples were not positive in the
mouse DNA control PCRs. Thus, although our single mtDNA
positive sample confirmed that mouse DNA contamination can
occur in blood samples from humans [8,9,10,12,32] it did not
change our XMRV detection result.
Further control PCRs
The sensitivities of the mouse mtDNA QPCR and IAP PCR
were tested by using serial 10-fold dilutions of mouse (C3H and
Balb/c) DNA. Both PCRs could detect mitochondrial DNA at a
quantity corresponding to 0.1–1 genome copy per PCR reaction.
There are around 10 000 mitochondrial DNA copies per mouse
cell [31]. The mouse genome (mm8 assembly) contains 3361 more
or less complete IAP proviruses (data from RetroBank [20]), and
probably an at least tenfold higher amount of IAP LTRs. The IAP
PCR was targeted to IAP LTRs. Given this high target frequency,
these mouse DNA tests must have had a roughly 1000- fold lower
sensitivity for mouse DNA than the XMRV/HMRV PCRs (gag
and env RTQPCR, and nested gag PCR; see above) which could
detect 1–10 copies per PCR reaction. The mitochondrial DNA
PCR was positive in one of eleven tested blood donor sera and in
none of two tested PBMC samples. The IAP PCR was positive in
none of eleven tested blood donor sera and in none of two PBMC
samples. Thus, we got evidence for mouse DNA contamination in
only 1 of 13 tested samples. Finally, the nested gag PCR of Lo et al
[3] was not positive in 11 tested blood donor sera and in none of
two tested PBMC samples. These control PCRs were used only for
Discussion
ME/CFS is a common disease [33]. It is often debilitating, but
despite several decades of research its clinical manifestations still
need to be more studied. All information which can contribute to
the understanding of this disease is important.
The retroviruses which we study occur both in exogenous and
endogenous form. The nonidentity between the human-derived
XMRV and its closest relatives in the mouse genome is 5–7%,
according to a BLAST search performed by JB (unpublished).
Assuming that mice were the source of XMRV (a likely
supposition) this small deviation, and the known high rate of
variation in exogenous retroviruses, indicates a rather recent
transmission to humans. Alternatively, all XMRV findings are
due to contamination from a common source. The design of tests
(PCR and serology) for infection with these viruses is highly
dependent upon information regarding the possible spectrum of
target viral sequences. A valuable resource is the not yet publicly
available ‘‘RetroBank’’ [20]. It is based on the computer program
Figure 5. Test of detection range of the env RTQPCR using synthetic target sequences. Alignment of primer (mauve and red) and probe
(green) target sequences with a synthetic PCR target sequence used to evaluate approximate detection width of the env QPCR. The detection limit
was determined by running PCR with tenfold dilutions giving 1–100 000 molecules per PCR reaction. The lowest positive dilution in the series, in
molecules per PCR reaction of the respective constructs, is written to the right of the alignment. The number of mismatches (underlined) between
forward primer, probe and reverse primer, respectively, is shown in the far right. chr13_68140880 is a group G2 MERV.
doi:10.1371/journal.pone.0024602.g005
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Figure 6. Histograms based on the histone 3.3 RTQPCR results for the three sample sets are shown. A. Data from PBMCs of ME/CFS/FM
patients. B. Data from plasma of ME/CFS/FM/patients. C. Data from blood donor sera.
doi:10.1371/journal.pone.0024602.g006
were retested with the nested gag PCR. An explanation could be
that the nested gag PCR was not quite as sensitive as the
RTQPCRs, which reached sensitivities of 1–10 target DNA
copies, or that retroviral NA was somewhat degraded during
subsequent freezing and thawing of the sample. The amplification
range predictions indicate that the MLV related retroviral
sequences, which are the ones reported in ME patients [2,3],
should be detectable with the gag and env RTQPCRs, whereas the
INT RTQPCRs should be confined to the most XMRV-like
targets, and should be less prone to false positivity due to mouse
DNA contamination.
It should also be emphasized that a substantial portion of the
300 high scoring murine gammaretroviral proviruses belong to the
here defined murine gammaretroviral groups G1 and G2. Both of
these contain some proviruses which are completely intact and are
strong candidates for being infectious. None of these should be
detectable with current PCRs. Thus, we may not have seen the
entire range of murine gammaretroviruses with zoonotic potential
for humans.
RetroTectorß [21,34]. The prototype of RetroBank currently
contains c:a 40 000 retroviral sequences from 30 vertebrate
genomes. The availability of this rich sequence source allowed us
to evaluate current XMRV/HMRV nucleic acid based tests for
expected range of retroviral detection. The result indicated that
the gag and env RTQPCRs described here, as well as the nested
gag PCR [3] should be able to detect most retroviruses related to
MLVs in group G3 in the tree in Figures 1 and 3, i.e. they should
have a similar detection range. The detection range of the INT
RTQPCR [35] seems to be much more narrow, which fits with
the absence of amplification from mouse DNA with this
RTQPCR. Nevertheless, the INT RTQPCR did not give a
positive result, according to our criteria of repeatability with
several different XMRV/HMRV specific PCRs. However, we
got two initially weakly reactive results in two samples. The
reason for the weakly reactive INT RTQPCR results is unknown.
Despite that the nested gag PCR [3] should have a similar
detection range as both gag and env RTQPCRs it did not become
positive when samples weakly reactive with these RTQPCRs
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MERV G3: In Mouse Genome but Not in Patients
Table 1. Summary of PCR results.
Category
Screening RTQPCR
N
Other PCRs
N
Other confirmatory assays
Plasma
INT (+), env 2, gag 2
1
nested gag PCR 2, mtQPCR 2, IAP1
PCR2
Plasma
INT 2, env 2, gag (+)
1
nested gag PCR 2, mtQPCR 2, IAP1
PCR2
1 contamination*
nested gag PCR 2, mtQPCR 2, IAP1
PCR2
No confirmatory sequencing done
nested gag PCR +, mtQPCR 2, IAP1
PCR 2
11 contamination*
nested gag PCR 2, mtQPCR +, IAP
PCR 2
1
nested gag PCR 2, mtQPCR 2, IAP
PCR2
9
ME+FM patients
(49 plasma, 85 PBMC)
Plasma
INT 2, env 2, gag 2
47
PBMC
INT 2, env 2, gag (+)
1
PBMC
INT 2, env 2, gag 2
84
Blood donors
(168 sera)
Serum
INT (+), env 2, gag 2
1
Serum
INT 2, env 2, gag (+)
11
Serum
INT 2, env 2, gag 2
156
(+) means weak and not repeatable positive.
*Contamination from synthetic target DNA proven by sequencing.
doi:10.1371/journal.pone.0024602.t001
distinguish true from false positivity due to contamination by
sequencing. The third case, contamination with mouse DNA, was
tested using the mouse mtDNA PCR and the IAP PCR. Likewise,
MLV-like proviruses, high and low scoring, predicted to be
detectable with most of the XMRV/HMRV PCRs, but not the
INT RTQPCR which is rather strictly XMRV specific, occur in
around 500 copies in the mouse genome (JB, data not shown).
Therefore, even a slight mouse DNA contamination in samples
subjected to XMRV/HMRV PCRs could cause a false positivity.
Biologicals may contain much vertebrate DNA, which in its turn
contains thousands of ERVs, possibly confounding sensitive and
broadly targeted PCRs. Heparin, for example, contains DNA from
the source animals (mostly pig and cow). The pig and cow
genomes do however not contain proviruses expected to react in
the PCRs used in this paper (JB, information from RetroBank
[20]). Moreover, patient samples (whole blood) analyzed in this
paper were collected in EDTA tubes (PBMCs) or plain glass tubes
(sera) and should not contain heparin. In a parallel titration, the
sensitivities of the mouse DNA PCRs were lower than that of the
gag and env RTQPCRs. The absence of XMRV/HMRV positive
results in our samples indicates that mouse DNA contamination
was a small problem in our samples. However, the results of our
bioinformatic search for MLV-like RV in vertebrate genomes, plus
our experience of this issue, allows a few comments. There are
similarities between the XMRV/HMRV and the ‘‘Human
Retrovirus 5’’ (HRV5) stories [36]. HRV5 is one of the so-called
‘‘rumor viruses’’ [37]. It turned out to be a rabbit retrotransposon
(RERV-H) whose DNA is abundant in rabbit sera [38]. The rabbit
genome contains around 700 copies of RERV-H [38]. Any
laboratory which handles rabbit sera is at risk of RERV-H
contamination. In analogy with this, a low level of mouse DNA
could be present in laboratory reagents or in the laboratory
environment [8,9,10,12,32]. However, non-template controls
should also be positive then. They were not. A source of mouse
The mouse retroviruses which have been reported to occur in
humans are potentially pathogenic. This kind of viruses can give
cancer (especially leukemia), encephalitis and immune deficiency
in mice and other natural hosts. It is therefore logical to investigate
their presence in patients with ME/CFS (encephalitis and immune
deficiency) and in cancer (prostate cancer). However, the absence
of a link to leukemia in humans is puzzling. Alternatively,
XMRV/HMRVs could be passenger viruses without disease
consequences. But the situation is interesting and should be
followed up. Because of the great variation in results, methodological optimization is a high priority.
Contamination is an omnipresent hazard whenever supersensitive tests are employed. The three screening PCRs used here can
detect 1–10 target molecules. There are three possible sources of
contamination, i.e. false positivity in the PCRs, which should be
considered, PCR amplimers, positive control DNA, and mouse
DNA. In the first case, the controls for PCR amplimer
contamination were non-template controls (PCR water; 1 to 4
samples per PCR round). None of them were positive. The three
RTQPCRs are non-overlapping, and therefore cannot contaminate for each other. In the second case, the control for positive
control DNA contamination, e.g. the XMRV VP62 clone, was
sequencing. We sequenced amplimers from the few weakly
reactive gag and INT RTQPCRs. The gag RTQPCR amplimers
had exactly the sequence of the synthetic positive control DNA for
the gag RTQPCR, shown in figure 4. It had the sequence of
XMRV VP62, but contained a characteristic 10 bp deletion.
These weak reactions must have been due to contamination with
this artificial DNA. Obviously, the number of non-template
controls per PCR round was too small to detect this low frequency
and low level of contamination. The sequences from the weakly
and not repeatably positive INT RTQPCRs were identical to the
target XMRV clone VP62 sequence for this PCR. The clone
sequence is identical in many XMRVs. This makes it hard to
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MERV G3: In Mouse Genome but Not in Patients
DNA in our laboratory is uncertain and unlikely. As shown here,
the likelihood of mouse DNA contamination causing reactions in
the INT RTQPCR, where we had a few weak reactions, is lower
than that of the other RTQPCRs.
Real-time PCR systems; gag RTQPCR with a minor groove
binding probe
The forward primer 59- AGAAGGTAGGAACCACCTAGT 39, Reverse-primer 59-TTTACCTTGGCIAAATTGGTG -39
(I = inosine), and the Probe: 59 - 6FAM-CGCCAGTTGCTCTTAGCGGGTCTCC MGB-NFQ-39, (6FAM, 6-carboxyfluorescein; MGB-NFQ is a minor groove binding tail with nonfluorescent quencher) (Applied Biosystems.Warrington, Cheshire, UK).
The PCR reaction contained 1 ml nucleic acid extract, 5 ml
5xQiagen OneStep RT-PCR Buffer from QIAGENH OneStep
RT-PCR kit (Catalog no. 210212; QIAGEN, Hilden, Germany),
1 ml dNTP mix (containing 10 mM of each dNTP), 300 nM
forward and reverse primers, 100 nM MGB-NFQ probe; RNasefree water; and 1 ml QIAGEN OneStep RT-PCR enzyme mix
(total volume 25 ml). The subsequent RT step (cDNA synthesis)
was performed at 50uC for 30 min, immediately followed by an
initial denaturation at 95uC for 15 min. A total of 45 cycles were
then performed, each consisting of a denaturation step at 95uC for
10 sec and an annealing-extension step in which the annealing
temperature was 50uC for 45 sec and extension at 60uC for 20 sec.
gag, env, INT and His3.3 RTQPCRs as well as the mitochondrial
DNA QPCR were performed using the Corbett Research
RotorGene Real Time Amplification system (RG 2000; Corbett
Research, Mortlake, NSW Australia). The RotorGeneTM software
version 6.4 (Corbett Research) was used for threshold selection
and standard curve interpolation to derive approximate RNA and
DNA concentrations relative to DNA standards. Artificial
synthetic targets for this and the other PCRs were ordered from
Biomers.net, Ulm, Germany.
Conclusions
The mouse genome contains three groups of high scoring
gammaretroviral proviruses. Some of them may have zoonotic
potential. The third group contains the highest proportion of
structurally intact proviruses, and is the target of most PCRs used
to detect XMRV/HMRV. Two new broadly targeted XMRV/
HMRV PCRs were developed. They were employed on samples
from ME/CFS patients and blood donors. False reactions due to
contamination with synthetic target DNA were encountered.
These could be classified as false by sequencing. The few
remaining reactions did not fulfill our criteria for positivity,
because they were not repeatable. The few weak and uncertain
PCR reactivities encountered by us are very different from the
high detection frequencies reported by others [2,39,40]. It is
possible that a higher amount of nucleic acid used per PCR could
have given a higher frequency of XMRV/HMRV detection.
However, many samples contained levels of nucleic acids similar to
those used in other studies [2,3,5,39,40]. Under the conditions
used by us, we could not corroborate that XMRV/HMRV is
frequent in Swedish ME/CFS/FM patients and blood donors.
Methods
Computer program for identification of conserved
portions in a set of aligned sequences
The program ConSort (JB, unpublished) displays the variation
and the number of contributing sequences are displayed. ConSort
was used for selection of target sequences for primers and probes
of the two new XMRV/HMRV-directed real-time PCRs
described in this paper.
env RTQPCR with MegaBeacon probe
The forward primer 59-GAGARGGCTACTGTGSYDMATGGG -39, Reverse-primer 59- CGGGTCARRGAGAACMGGGTC -39, and the XMRV_MegB2 Probe: 59- CAATCCCCTAGTCCTAGAATTCACTGACGCGGGTAAAAAtaggggattg-39
The MegaBeacon probe was labeled with the fluorescent
reporter dye JOE at the 59-end and the quenching Dabcyl at
the 39-end position (Eurogentec Seraing, Belgium). The underlined sequence at the 59 and 39 ends identifies the arm sequences
of the MegaBeacon that is the stem, (10 bp). The 10 nucleotides at
the 39 end (taggggattg) are not complementary to the target
(Information S4). The PCR reaction contained 1 ml nucleic acid
extract, 12.5 ml 26 RT-PCR Step RT-PCR kit for Probes
(BioRad, Sundbyberg, Sweden), 400 nM forward and reverse
primers, 200 nM MegaBeacon probe; nuclease-free water; 0.5 ml
iScript reverse transcriptase enzyme (total volume 25 ml). The
subsequent RT step (cDNA synthesis) was performed at 50uC for
30 min, immediately followed by an initial denaturation at 95uC
for 15 min. A total of 45 cycles were then performed, each
consisting of a denaturation step at 95uC for 30 sec and an
annealing-extension step in which the annealing temperature was
46uC for 45 sec and extension at 72uC for 20 sec. Fluorescence
intensity was measured at the end of the extension step in each
cycle.
Gammaretroviruses of C57BL/6J mice (genome assembly
mm8)
This assembly was analyzed with the RetroTectorß system
(ReTe) [21]. The results were stored in a prototype version of
RetroBankß [20]. Gammaretroviruslike proviruses which scored
more than 2000 were selected. This yielded 300 proviruses.
Examples of the most structurally intact gammaretroviruslike
proviruses from the cow, pig, guinea pig, rabbit, tupaia, marmoset,
rhesus, baboon, chimpanzee and human genomes were also
included as references. Alignments were made using ClustalW [41]
version 1.83, and MUSCLE [42,43]
Phylogenetic trees were either the Neighbour-Joining (NJ) guide
trees from ClustalW, or Minimum Evolution (ME) and Maximum
Likelihood (ML) trees, with bootstrapping where appropriate,
embodied in the MEGA [44] program suite.
Bioinformatic assessment of amplification range of the
QPCRs
The forward and reverse primers, and the probes, were aligned to
the gag and env alignments of the 300 gammaretroviruslike
proviruses. The degree of fit between target sequence and primers
and probe was calculated using a modified version of the NucZip
algorithm [28]. The predicted detection ability (PDA) was
calculated using the product of primer scores, and probe score.
PDAs were displayed for each member of the aligned sequences,
and mapped onto a Neighbor-Joining (NJ) tree of gag, integrase and
env nucleotide sequences targeted by the respective PCR system.
PLoS ONE | www.plosone.org
INT RTQPCR
This was performed as in the paper of Schlaberg et al. [35], with
slight modifications. Briefly, the reaction mix consisted of 10 ml 26
iScript RT-PCR reaction mix buffer from iScriptTM One-Step
RT-PCR kit for Probes (BioRad, Sundbyberg, Sweden), 900 nM
XMRV4552F (59-CGAGAGGCAGCCATGAAGG-39; forward
primer), 450 nM XMRV4653R (59-GAGATCTGTTTCGGTGTAATGGAAA-39; reverse primer1), 450 nM XMRV4673R (5910
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MERV G3: In Mouse Genome but Not in Patients
CCCAGTTCCCGTAGTCTTTTGAG-39; reverse primer2), 250 nM
XMRV 4572MGB probe (59- 6FAM - AGTTCTAGAAACCTCTACACTC MGB-NFQ -39) (like for the gag RTQPCR, MGBNFQ is a minor grove binder with nonfluorescent quencher)
(Applied Biosystems,Warrington, Cheshire, UK); nuclease-free
water; 0.5 ml iScript reverse transcriptase enzyme and 6 ml of
template per PCR reaction in a total reaction volume of 20 ml.
The subsequent RT step (cDNA synthesis) was performed at 50uC
for 30 min, immediately followed by an initial denaturation at
95uC for 15 min. A total of 45 cycles were then performed, each
consisting of a denaturation step at 95uC for 10 sec and an
annealing step at 60uC for 45 sec.
Histone 3.3 (His3.3) RTQPCR
A reverse transcription real-time histone 3.3 RNA and DNA
QPCR [45] was always run in parallel with the other PCRs to
ensure amplifiability of the samples with slight modifications.
Briefly, the reaction mix (25 ml) consisted of 1 ml nucleic acid
extract, 12.5 ml 26 RT-PCR Step RT-PCR kit for Probes
(BioRad), 200 nM histone forward primers 59-CCTCTACTGGAGGGGTGAAGAA- 39; 200 nM histone reverse primers 59TGCCTCCTGCAAAGCACCGATA- 39; 200 nM Probe: 6FAM
-CTCTGGAAGCGCAGATCTGTTTTAAAGTCCT- MGBNFQ-39, (Applied Biosystems.Warrington, Cheshire, UK); nuclease-free water and 0.5 ml iScript reverse transcriptase enzyme. The
subsequent RT step (cDNA synthesis) was performed at 50uC for
30 min, immediately followed by an initial denaturation at 95uC
for 15 min. A total of 55 cycles were then performed, each
consisting of a denaturation step at 95uC for 15 sec and an
annealing step at 54uC for 60 sec. Serial dilutions of a histone 3.3
plasmid containing 106–100 copies per PCR reaction were used in
the experiment as quantitative standards. Results were expressed
as ‘‘Histone 3.3 equivalents’’ (HIEQ). In twenty samples, HIEQ
were correlated with DNA concentration determined with a
NanoDropH Spectrophotometer (NanoDrop Technologies inc.
Wilmington, USA). On average, 716 HIEQ/uL corresponded to
1 ng/uL of DNA determined by photometry.
XMRV/MLV gag Nested PCR
A wide range of XMRV-related mouse viruses (HMRV) were
detected in ME patients [3]. In the first round PCR, the PCR
reaction contained 1 ml nucleic acid extract (corresponding to up to
1017 ng of total cellular nucleic acid/reaction as judged by the
histone 3.3 RTQPCR result) in total reaction volume, 20 mL, 16
Taq buffer, 2.5 mM MgCl2, 0.2 mM dNTP, 0.25 pmol/mL of
419F 59-ATCAGTTAACCTACCCGAGTCGGAC-39 primer,
0.25 pmol/mL of 1154R 59-GCCGCCTCTTCTTCATTGTTCTC-39 primer (outer primers) (biomers.net, Ulm, Germany),
and 0.5 units of AmpliTaq Gold Taq (Applied Biosystems.Foster
City, CA). For the second round PCR, the PCR reaction contained
2 mL of round 1 PCR product 16 Taq buffer, 2.5 mM MgCl2,
0.2 mM dNTP, 0.25 pmol/mL of NP116 59-CATGGGACAGACCGTAACTACC-39 primer or GAG-I-F 59-TCTCGAGATCATGGGACAGA-39 primer, 0.25 pmol/mL of NP117 59-GCAGATCGGGACGGAGGTTG-39 primer or GAG-I-R 59AGAGGGTAAGGGCAGGGTAA-39 primer, and 0.5 units of.
The cycles for both PCRs were 4 min at 94uC (1 min at 94uC,
1 min at 57uC, 1 min at 72uC)640 cycles and 10 min at 72uC.
Following amplification, 5 ml of PCR-product was separated by
electrophoresis on 2% agarose gel. The first-round PCR amplifies
a fragment of ,730 bp from the gag gene. The second-round PCR
gives a product of 413 bp, using the GAG-I-F and GAG-I-R
primers, or a product of 380 bp using the NP116 and NP117
primers.
Sequencing of PCR amplimers
PCR amplimers were purified by QIAquick PCR Purification kit
(QIAGEN, Hilden, Germany) and cloned using the TOPOTM TA
Cloning Kit (Invitrogen, Stockholm, Sweden). The plasmids DNA
were isolated by using QIAprepH Spin Miniprep Kit (QIAGEN,
Hilden, Germany). The concentration of plasmid DNA was
quantified by using the NanoDropH Spectrophotometer and then
using with M13 primers and the fluorescent dye terminator
reagents, ABI PRISMH Big DyeTM Terminator v3.1 Cycle
Sequencing kit (Applied Biosystems, Foster City, CA) and on an
ABI PRISMH 310 genetic analyzer according to the manufacturer’s
recommendations (Applied Biosystems, Foster City, CA, USA).
Mouse mitochondrial DNA (mtDNA) QPCR
Mouse DNA contamination in reagents and patient samples is a
possibility [9,10]. This control PCR was used with samples
reactive in any of the gammaretrovirus-targeted PCRs. The
primer and probe sequences for murine mitochondrial cytochrome oxidase, cox2, were kindly provided by dr William Switzer,
Centers for Disease Control, Atlanta, USA. The reaction mix
consisted of 12.5 ml 26 iScript RT-PCR reaction mix buffer from
iScriptTM One-Step RT-PCR kit for Probes (BioRad, Sundbyberg, Sweden), 320 nM MCox2-F2 (59-TTCTACCAGCTGTAATCCTTA-39), 320 nM MCox2-R1 (59- GTTTTAGGTCGTTTGTTGGGAT-39) primers, and 160 nM MCox2-PR1 (59FAM-CGTAGCTTCAGTATCATTGGTGCCCTATGGT-BHQ
-39),160 nM MCox2-P1 (59- FAM-TTGCTCTCCCCTCTCTACGCATTCTA-BHQ -39) probes (biomers.net, Ulm, Germany); nuclease-free water, 5 ml of template per PCR reaction in a
total reaction volume of 25 ml. Thermocycling conditions were
95uC for 10 min, followed by 55 cycles of 95uC for 30 sec and
60uC for 30 sec. Serial 10-fold dilutions of Balb/c and C3H/HeJ
9384 mouse DNA extracts were used to validate the assay.
Control samples for the XMRV/HMRV PCRs
Positive controls for gag, INT and env PCRs consisted of nucleic
acid extracts from supernatants of the 22Rv1 XMRV-producing
prostate cancer cell line (ATCC CRL 2505), and 3 DNA extracts
from ME patient PBMC samples found positive for XMRV at the
Whittemore-Peterson Institute (WPI) in Reno, Arizona (a kind gift
from dr Judy Mikovits). Two of them were weakly positive in the
INT RTQPCR, with Ct 41.8 and 37.7, respectively). A positive
control for gag, env and mouse mtDNA QPCRs was DNA
extracted from an adult female Balb/c laboratory mouse (Charles
River, Denmark) (a gift from dr Ylva Molin, Uppsala; The DNA
was obtained from a laboratory mouse reared in Uppsala. The
mouse was reared according to the recommendations in ‘‘Guide
for the Care and Use of Laboratory Animals’’ of the Swedish
National Board for Laboratory Animals (CFN). The rearing and
taking of samples from the mouse was approved (C127/4) by the
local Ethical Committee for Experimental Use at the Faculty of
Medicine, Uppsala University.)
The INT QPCR did not amplify from these mouse DNAs (as
reported earlier by dr Singh [35]). Negative (non-template) control
was DEPC-Treated Water (Ambion, INC. Austin, USA) and from
nuclease-free water included in both the iScriptTM One-Step RTPCR kit for Probes (BioRad, Sundbyberg, Sweden) and the
QIAGENH OneStep RT-PCR kit (QIAGEN, Hilden, Germany).
PLoS ONE | www.plosone.org
PCR assay for Mouse intercisternal A-type particle (IAP)
LTR DNA
The primer sequences, which are targeted to the long terminal
repeats of the retrotransposon intracisternal type A particle, were
11
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MERV G3: In Mouse Genome but Not in Patients
kindly provided by dr Oya Cingöz (Tufts, Massachussetts, USA).
The PCR reaction was carried out in an total volume of 50 mL
containing 1 ml nucleic acid extract, 16PCR buffer minus Mg,
1.5 mM MgCl2, 0.2 mM dNTP mixture, 200 nM of IAP-F 59ATAATCTGCGCATGAGCCAAGG -39 forward primer,
200 nM of IAP-R 59- AGGAAGAACACCACAGACCAGA -39
reverse primer (Thermo Fisher Scientific, Ulm, Germany),
nuclease-free water and 1 U Taq DNA polymerase (Invitrogen,
Lidingö, Sweden). Thermocycling conditions were 40 cycles of
95uC for 30 sec, 59uC for 30 sec and 72uC for 30 sec. The
expected and observed product sizes were 235–350 bp.
Gothenburg (Dnr 680-09), which allowed the samples reported
here to be taken.
Blood donor samples
Sera from 168 consecutive anonymous blood donors were
obtained from the blood bank at Uppsala Academic Hospital,
Sweden.
The blood donors gave written consent to the use of their serum
for analysis of blood-borne viruses according to the routine of the
Academic Hospital in Uppsala, and a general permit for this
purpose from the Ethical Committee of the Medical Faculty of the
Uppsala University (2004).
RNA and DNA extraction
The total nucleic acid was extracted from PBMCs of EDTA
blood of ME/CFS/FM patients and sera from blood donors as
described by the manufacturer (EasyMagH, bioMérieux, Boxtel,
Netherlands). The samples were eluted in 60 ml and stored at
270uC. For samples from ME/CFS patients from Gothenburg,
whole blood samples were obtained in CPT tubes (Becton
Dickinson, Stockholm, Sweden), and centrifuged as specified by
the manufacturer, at 1700 g for 20 min at room temperature. The
PBMC fraction (1 ml) was then taken, and 500 ml of it was used for
nucleic acid extraction with the EasyMag. Plasma from 49 out of
the 85 ME/CFS/FM patients was analyzed. Two hundred ml of it
were used for nucleic acid extraction with the EasyMag.
Criteria for XMRV/HMRV PCR positivity
Samples were interpreted as ‘‘positive’’ if repeatable signals with
at least two different XMRV/HMRV PCRs were obtained.
Samples were interpreted as ‘‘weakly reactive’’ if they were
reactive only once in one of the three screening RTQPCRs.
Supporting Information
Table S1 Detailed list of the members of the G1–G3
groups.
(PDF)
Information S1 Phylogenetic trees supporting the G1–
G3 groups, and the G3 subgroups (poly-, modified polyand xenotropic), as well as ecotropic, MERVs. Relationship to other MLV-related gammaretroviruses.
(PDF)
Efforts to reduce the likelihood of PCR contamination
To avoid false positive results due to DNA or RNA
contamination filtered pipette tips, PCR hoods with ultraviolet
light and separate rooms for PCR preparation and product
analysis were used. One to four negative (non-template) controls
were also included in every experiment. To detect any mouse
DNA contamination of the extraction reagents, 300 ml of
NucliSensH Lysis Buffer was blindly extracted in eight samples.
They came out negative in the gag RTQPCR and the mouse DNA
PCR.
Information S2 Xenotropic cell culture contaminating
retroviruses and the uniqueness of 22RV1/XMRV.
(DOC)
Clinical samples
Information S4 Development of the gag and env
Information S3 The G1–G3 groups. Properties and
consensus sequences.
(DOC)
RTQPCR; Evolutionary conservation of target sequences.
(PDF)
The 85 patients included 48 patients with the diagnoses ME/
CFS according to the Canadian criteria [46] and 30 patients with
both ME/CFS and FM diagnosis. Seven patients only fulfilling the
criteria for FM were also included. The FM diagnosis was
according to ACR classification [47]. IBS was diagnosed in 40% of
the total group of 85 patients with no significant difference in the
subgroups. All patients were rated by the FibroFatigue scale [48].
The mean score was 4169 points indicating moderate to severe
degree of disorder. The total variance of the scale is 0–72.
Diagnosis was made by three doctors, all M.D. and PhD, well
trained in the use of the rating scale and in the diagnosis of the
disorders. RNA and DNA were extracted from peripheral blood
mononuclear cell (PBMC) of the 85 ME/CFS/FM patients. RNA
and DNA were also extracted from plasma of 49 ME/CFS/FM
patients.
All patients from the Gothenburg study gave written consent
according to a permit from the Ethical Committee of University of
Acknowledgments
The expert help of Akofa MacKwashie and Bengt Kallin with the nucleic
acid extraction robot is gratefully acknowledged. We thank Bengt Kallin
and Kåre Bondeson for discussions regarding the nucleic acid extraction
conditions, and the PCR results.
This paper is dedicated to the memory of Xingwu Shao.
Author Contributions
Conceived and designed the experiments: JB AE XS SM JMU AW.
Performed the experiments: AE XS SG JMU AW. Analyzed the data: AE
XS JB SM JMU AW MM OZ CGG. Contributed reagents/materials/
analysis tools: JB CÖ CGG MM OZ. Wrote the paper: JB AE CGG JM
CÖ. Arranged funding: JB CGG.
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