ME -The Cat that Caught the Mice

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ME Advocacy

The 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

sequences.

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.

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.

* 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.

Source

Department of Human Genetics, Center of Human

and Molecular Biology, Medical Faculty, University

of Saarland, 66421 Homburg, Germany.

Abstract

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 M. 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 human retrovirus.

Photo Fig.

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 this

study.

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.

REFERENCES

1 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. (http://bit.ly/fstKac)

2 NCI. 2011. Cancer Research Now: Investigating

XMRV. Youtube. (http://bit.ly/PhcttV)

3 Paprotka et al. 2011. Recombinant Origin of

the Retrovirus XMRV. Science. 333, 97-101.

(http://bit.ly/Phd4f5)

4 Mayer et al. 2012. Comparing PreXMRV-2 gag

sequence diversity in laboratory and wild mice

using deep sequencing. Virus Res.

(http://bit.ly/M7YhVU)

5 Cingoz et al. 2012. Characterization, Mapping

and Distribution of the Two XMRV Parental

Proviruses. JVI. DOI:10.1128/JVI.06022-11.

(http://bit.ly/PhdWjK)

6 Lombardi et al. 2009. Detection of an Infectious

Retrovirus, XMRV, in Blood Cells of Patients

with Chronic Fatigue Syndrome. Science. 326,

585-589. (http://bit.ly/g7Tp7G)

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