The cat that caught the mice
see http://me-advocacy.com/Cat_that_caught_the_mice.htmlThe cat that
caught the mice
Dr Coffin and Dr Pathak first voiced their opinion on the origin of XMRV
at CROI conference 2011 (1). Later they released a video (2) which
recounted how they had traced the birth of the virus to one cell line,
called 22Rv1, and how they had also miraculously identified two parental
viruses to XMRV (PreXMRV-1 and PreXMRV-2). It was a fantastical tale
and had all the hallmarks of wondrous discovery that only the keenest
most focused minds could have identified, or at least this was the
impression many would have been given from the publicity surrounding the
paper, Paprotka et al. 2011 (3).
On further investigation the experiments in the paper, 'Recombinant
origin of the retrovirus XMRV' (3), could not have supported these
claims and the sequence uploaded into the GenBank which was said to have
come from the 22Rv1 cell line, and was named the "consensus" XMRV
sequence, did not include an env gene. It had also been discovered that
XMRV was not the viruses that had been detected in the human population
by various scientists. They were actually XMRV-like.
Earlier this week scientists from Germany published a study, Mayer et
al. 2012 (4), looking for one of the viruses which Paprotka et al. had
claimed was an ancestral virus to XMRV. Their research directly
contradicts what Paprotka et al. (3) and Cingoz et al. 2011 (5) had
proposed for the origin of this ancestral virus and the human XMRV-like
A short recap of Paprotka et al. (3) and Cingoz et al. (5). is provided
at the bottom of this article. The main points are given here in bullet
form as they relate to the German study.
No wild mice were screened in either paper.Only PCR was used in both
studies.45 Lab mice and 44 wild derived lab mice were negative for XMRV
in Paprotka et al.15 lab mice screened for PreXMRV-1 and -2 in Paprotka
(no wild derived lab mice). Several strains were positive.48 lab strains
and 46 wild derived lab strains were screened for PreXMRV-1 and -2 in
Cingoz et al. Several strains were positive.22Rv1 cell line created
using nude mice.So called PreXMRV-1 and -2 viruses found integrated into
hairy mice and not nude mice in Cingoz et al.PreXMRV-1 and PreXMRV-2
were claimed to be endogenous mouse viruses in Paprotka et al.
RESULTS OF THE GERMAN STUDY
Virus Res. 2012 Jul 4. [Epub ahead of print]
Comparing PreXMRV-2 gag sequence diversity in laboratory and wild mice
using deep sequencing.
Mayer J, Mazzoni CJ, Greenwood AD.
Department of Human Genetics, Center of Human and Molecular Biology,
Medical Faculty, University of Saarland, 66421 Homburg, Germany.
It has recently been reported that the xenotropic murine leukemia
virus-related virus (XMRV) derives from a laboratory recombinant.
However, sequences with characteristics of the 5' half of XMRV (termed
PreXMRV-2) have been identified in several laboratory mouse genomes and
cell lines suggesting parts of the XMRV genome exist as naturally
occurring retroviruses in mice. We compare here PreXMRV-2 gag sequence
diversity in mice to that of reported XMRV-like sequences by testing a
panel of wild mouse and common inbred laboratory mouse strain genomic
DNAs and by using high throughput amplicon sequencing. Sequences with
features typical of previously reported PreXMRV-2 sequences, among them
a 24 nt deletion, were repeatedly identified in different wild mice and
inbred mouse strains within a high background of non-XMRV-like
sequences. However, Sanger sequencing of clones from amplicons failed to
retrieve such sequences effectively. Phylogenetic analysis suggests that
PreXMRV-2 gag sequences from mice, cell lines and patient samples belong
to the same evolutionarily young clade and that such sequences are
diverse and widespread within Mus musculus domesticus and laboratory
mice derived from this species. No evidence of PreXMRV-2 like gag
sequences could be obtained outside of the Mus musculus lineage. The
results suggest that accurate determination of presence, absence and
relationships of specific murine retroviral strains benefit greatly from
deep sequencing analysis.
Mayer et al. compared the diversity of PreXMRV-2 and XMRV-like sequences
in mice, both inbred strains and wild strains, and in a number of other
rodents, using both low throughput assays (PCR) and high throughput
assays (next generation sequencing). In comparison to PCR, next
generation sequencing (NGS) produces millions more sequences of a virus.
Making it a far better approach for detecting low level infection by a
specific virus, than antiquated PCR techniques. Surprisingly no study
had previously used NGS to chart the diversity of MLVs within mice or
using multiple individual mice.
According to the authors, the PreXMRV-2 virus was chosen as the
experiments could be constructed around the 24 nucleotide deletion in
the gag region of the virus. For those who are not aware, PreXMRV-2 is a
polytropic virus that is 99.9% similar in the gag and partial pol
regions to XMRV, which also has a 24 nucleotide deletion in its gag
region. It is also important to note that the 67% positivity figure
from Lombardi et al. 2009 (6) was obtained using an assay targeting the
gag region of VP35, a virus with a high similarity (99.8%) in the gag
region to PreXMRV-2. Of the two primer pairs used in this study the
second longer set are designed to detect the gag region of the VP35
Mayer et al. discovered that where NGS sequencing could detect MLV
viruses, PCR used in the Paprotka and Cingoz papers would generally
detect nothing. Two reasons were suggested for this. The low copy
number of the viruses and the partial deletion in the gag region of the
virus interfering with the specificity of the assays used.
The authors also highlighted how Cingoz et al. had claimed to have
identified XMRV-like sequences in various mice, but how they had
actually not attempted to sequence the products amplified by PCR, making
identification of the viruses merely an assumption.
Also, in contradiction to the findings of Paprotka and Cingoz using PCR,
Mayer et al. also found that when using NGS PreXMRV-2 has sequence
diversity in wild and inbred mice. This indicates that PreXMRV-2 cannot
be an endogenous mouse virus.
",human derived, cell line derived and mouse DNA derived XMRV-like
sequences showed poor tree structure, high diversity in the number of
lineages and did not cluster by DNA source…"
Their phylogenetic analysis also indicated that most of the sequences
containing the 24 nucleotide deletion from mice, cell line and human
tissue, as well as sequences without the deletion, were all derived from
an unknown clade, which contained a mix of polytropic and xenotropic
sequences. Several sequences were similar but diverged from the
PreXMRV-2 virus after the 24 nucleotide deletion and were closer to
Xenotropic murine virus sequences. An indication that these were
recombinant sequences combining PreXMRV-2 and a xenotropic virus. A
common occurrence with viruses that have high recombination rates. Due
to the high similarity of the sequences the clade was also thought to be
young. In gamma retroviral terms this would be a few hundred years.
"The PreXMRV-2 virus likely originated recently in wild populations of
M. domesticus and may not be present in all populations."
From the mice tested they found that PreXMRV-2 likely originated in Mus
musculus domesticus (western European house mouse). That several lab
mice and inbred lab strains were also infected. Which would be
expected, as the majority of inbread lab mice are derived from M. m.
domesticus and some would inherit the PreXMRV-2 gag.
The authors also tested a museum sample of a wild mouse (M. m.
domesticus) captured in Ann Arbor Michigan, USA, in 1906. The PCR
products from this sample were correct for this to be infected with a
virus from the PreXMRV-2 family clade, but due to the age of the sample
and subsequent degradation of the DNA they were unable to sequence.
Leading them to state that:
"we cannot conclude that PreXMRV-2 like sequences are absent in the
U.S. wild house mouse population. "
The authors suggest that PreXMRV-2 gag may be broadly present among
other various susbspecies of Mus musculus which they had not tested in
They concluded that the virus was not present at a high copy number in
mice and that consequently high-throughput (NGS) sequencing should
generally be employed when attempting to detect rare sequences of this
type. NGS also being readily used for detecting rare HIV-1 variants.
The authors concluded that:
"The presence of a distinct clade including XMRV, PreXMRV-2, cell
line-derived and mouse DNA-derived sequences suggests that the 24 nt
deletion and gag specific features are shared due to common ancestry and
not due to independent generation of the deletion or other polymorphisms
within this group."
Again the main conclusions of the study are as follows:
PreXMRV-2 has diversity. High-throughput sequencing will identify MLVs
which PCR cannot.PreXMRV-2 likely originated in M. m. domesticus.
IMPLICATIONS FOR HUMAN GAMMARETROVIRAL INFECTIONS
This research is the final nail in the coffin for the lab contamination
story for the ME and prostate cancer retroviruses. The viruses have not
been confined to the lab. It is highly probably that recombination
events with PreXMRV-2 are occurring all the time in the wild, with both
other polytropic and xenotropic MLVs. If anything this paper shows that
without full sequencing of the ME retroviruses and more prostate cancer
retroviruses, no claim can be made as to the variants and strains which
has been detected.
In addition, if MLVs cannot always be detected when using PCR, then how
can we expect researchers to correctly determine whether the same
viruses are infecting humans when they are using those inferior
techniques? The negative studies are obsolete.
Much is not known about these viruses, but we now know that
high-thoughput sequencing needs to be a prerequisite in further studies
to uncover the truth. Too much money and time has been lost chasing
viruses that can escape PCR detecting.
Paper # 91LB. Paprotka et al. 2011. XMRV Probably Originated through
Recombination between 2 Endogenous Murine Retroviruses during in vivo
Passage of a Human Prostate Cancer Xenograft. CROI.
NCI. 2011. Cancer Research Now: Investigating XMRV. Youtube.
Paprotka et al. 2011. Recombinant Origin of the Retrovirus XMRV.
Science. 333, 97-101.
Mayer et al. 2012. Comparing PreXMRV-2 gag sequence diversity in
laboratory and wild mice using deep sequencing. Virus Res.
Cingoz et al. 2012. Characterization, Mapping and Distribution of the
Two XMRV Parental Proviruses. JVI. DOI:10.1128/JVI.06022-11.
Lombardi et al. 2009. Detection of an Infectious Retrovirus, XMRV, in
Blood Cells of Patients with Chronic Fatigue Syndrome. Science. 326,
PAPROTKA ET AL. 2011 AND CINCOZ ET AL. 2011
The claim in Paprotka was simple. A linear series of cells originally
from a prostate cancer patient which had been passed through mice had
been analyzed with various tests. There were cells from early passages
and later passages through mice, and a third set of cells established
much later. They could not find XMRV in the earlier cells, but could in
both of the later cells. They also claimed that two endogenous viruses
(PreXMRV-1 and PreXMRV-2) were present in mice which in their words were
"likely to have been used" in creating the 22Rv1 cell line. Three
separate tests had been used to come to this conclusion.
The three tests were however used selectively on different cells and
mice, and each had a different limit of detection. The first two tests
were in combination used on all items but the later cells that were
passed through mice. Neither was ever established capable of detecting
XMRV at less than 2000 copies per 100 cells. The third test being the
most sensitive type of the three tests was only used to then screen the
later cells that had been passed through mice, but no data was provided
on the sensitivity of this test (in fact the test is not even named in
the paper) and those cells in particular were never shown to have been
from the same patient as all other cells tested.
Of the mice that were screened there were no wild mice included, only
several strains of lab mice which had been derived from wild mice and a
number of lab mice strains. None of the mice were those actually used
in the creation of the 22Rv1 cell line.
In summary the experiments within the study were not capable of
determining the origin of the virus was a single cell line. The two so
called parental viruses were also not shown to be present for this
magical recombination to have occurred. Finally the uploaded sequence
of the virus put into the NCBI database from the 22Rv1 cells was later
found to not include an envelop gene, and thus could not be claimed to
be the XMRV virus.
A later study from Dr Coffin (Cingoz et al. 2011), took the tale of the
two parental viruses further. There he claimed that the viruses were
integrated into the genome of various lab mice, but the mice were not
nude mice which were those used for the creation of the 22Rv1 cell line.
15 July 2012