Full Text: ME/CFS -Xenotropic & polytropic murine leukemia virus-related sequences not detected in majority of patients

[Guest guest]

Several hours ago I posted the abstract

of:

Xenotropic and polytropic

murine leukemia virus-related

sequences are not detected in

the majority of patients with

chronic fatigue syndrome

Reference: plain text version at Co-Cure:

http://bit.ly/QiJhmI

Below you will find an easy-readable full

text version and an attachment in pdf

format of the original study (private

members), which can also been found

at: http://bit.ly/Qik3ES

~jvr

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NEW MICROBIOLOGICA, 35, 341-344,

2012

Xenotropic and polytropic

murine leukemia virus-related

sequences are not detected in

the majority of patients with

chronic fatigue syndrome

Stefania Paolucci1, Piralla1,

Cinzia Zanello1, Lorenzo Minoli2, Fausto

Baldanti1 1S.S. Virologia Molecolare,

S.C. Virologia e Microbiologia,

Fondazione IRCCS Policlinico San

Matteo, Pavia, Italy; 2Clinica Malattie

Infettive, Fondazione IRCCS Policlinico

San Matteo, Pavia, Italy

Corresponding author Fausto Baldanti

S.S. Virologia Molecolare S.C. Virologia e

Microbiologia Fondazione IRCCS

Policlinico San Matteo Via Taramelli, 5 -

27100 Pavia, Italy E-mail:

f.baldanti@...

Received February 9, 2012 Accepted

April 24, 2012

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SUMMARY

XMRV and polytropic MLV-related virus

have been controversially associated

with chronic fatigue syndrome (CFS).

Subsequent reports failed to detect

XMRV and MLV-related virus in CFS

patients, and the previous results have

been interpreted as a massive

laboratory contamination by mouse

DNA sequences.

Among 12 sequential CFS patients, two

were positive for XMRV/MLV sequences.

In contrast, 40 selected control subjects

were negative.

CSF patients and controls were negative

for mitochondrial mouse-specific DNA

sequences.

These findings do not confirm the high

frequency of MLV-related viruses

infection in CFS patients, but also

contrast the widespread laboratory

contamination previously suggested.

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Chronic fatigue syndrome (CFS) is an

uncommon clinical condition of unknown

etiology characterized by persistent

fatigue in association with malaise,

multiple joint and muscle pain,

unrefreshing sleep and severe mental

and physical exhaustion.

Recently, the xenotropic murine leukemia

virus (MLV)-related virus (XMRV)

genome sequence was identified in

prostate tissue of 40% of patients with

prostate cancer (Urisman et al., 2006)

and in peripheral blood mononuclear

cells (PBMC) of 67% of patients with

CFS (Lombardi et al., 2009).

The XMRV sequences detected in these

patients were almost identical, whereas

they differed from those of the related

ecotropic MLV detected in prostate

cancer patients (Urisman et al., 2006).

More recently, an MLV-related virus,

more closely related to the polytropic

MLV than to XMRV, was identified in

CFS patients (Lo et al., 2010).

However, subsequent studies failed to

detect XMRV and MLV-related virus in

tissues or blood samples of CFS patients

(Erlwein et al., 2010; Switzer et al.,

2010; et al., 2011; Shin et al.,

2011), and the previous positive findings

were interpreted as the result of

contamination by a laboratory-derived

virus (Paprotka et al., 2011).

Finally, the original papers by Lombardi

and Lo (Lombardi et al., 2009; Lo et al.,

2010) had been retracted and no

correlation between the presence of

XMRV/MLV and CFS has been

recognized (Karafin and Stramer, 2012).

However, in several papers denying the

association between XMRV/MLV and

human diseases, a subset of samples

showed positive PCR (Arredondo et al.,

2011; et al., 2011) or serologic

(Arredondo et al., 2011; Qiu et al.,

2012) signals.

During 2010 the presence of XMRV and

polytropic MLV-related provirus was

investigated in 12 sequential CFS

patients.

As a control, matching numbers of

randomly selected individuals with

different clinical conditions

(HIV-infected individuals, n=10,

transplant recipients, n=10,

HCV-positive patients, n=10, and

healthy subjects, n=10) were analyzed

n parallel during the same period.

Following digestion of 10_6 (10 to the

sixth) PBMC with proteinase K, DNA

was extracted using the easy MAG™

automatic extractor (Biomerieux, Lyon,

France).

ested PCR of the XMRV gag region was

performed using previously described

primer pairs (Urisman et al., 2006;

Lombardi et al., 2009).

A second XMRV gene was amplified in

parallel by a real-time PCR assay

detecting a fragment of the XMRV

integrase gene (Schlaberg et al., 2009).

The XMRV VP62 clone (obtained from the

NIH AIDS Research and Reference

Reagent Program, Rockville, MD, USA)

was used as a positive control. bPta

2-microglobulin was amplified in all

samples to normalize real-time PCR

results.

To prevent potential carry-over

contamination, samples were processed

in three separate rooms, multiple

negative controls were included in each

assay and real-time PCR assays were

performed using the TaqMan Universal

PCR Master Mix including the UNG

(Applied Biosystems, City, CA,

USA).

To exclude potential contamination by

mouse DNA, mousespecific mitochondrial

DNA sequences were searched for as

reported (Lo et al., 2010). PCR

amplicons were sequenced using the

BigDye Terminator Cycle Sequencing

Ready kit (Applied Biosystems,

City, CA).

Viral sequences were analyzed by the

Blast program (http://1.usa.gov/QiEwcT).

Multiple sequence alignment was

performed with the MEGA version 5.0

software for the phylogenetic analysis.

Two out of 12 (16.6%) CFS patients

were positive for gag PCR and integrase

PCR respectively.

In contrast, no evidence of XMRV or

other MLV-related viruses was detected

in blood cells from HIV-positive patients,

HCV-positive patients, transplant

recipients or healthy donors.

Nucleotide sequencing of the gag

amplicon from the first positive patient

(CFS Pavia-1) showed that the

retrovirus was more closely related to

the polytropic MLV rather than to XMRV

(identity 100% vs. 96%) (Figure 1A).

In particular, a specific deletion of seven

amino acids between codon 45 and

codon 51 of the gag gene observed in

our strain and five additional virus

strains reported in GeneBank

characterized the similarity of these

retroviruses to the polytropic

MLV-related virus.

Sequencing of the integrase gene

amplicon from the second positive

patient (CFS Pavia-2) confirmed the

similarity with the XMRV sequence

(Figure 1B).

The finding of two related, but different,

virus sequences (polytropic MLV and

XMRV) in two CFS patients, does not

confirm the high frequency of

MLV-related virus infection in CFS

patients reported in previous studies

(Lombardi et al., 2009).

On the other hand, our results are in

contrast with the reported massive and

widespread laboratory and reagents

contamination ( et al., 2010;

Oakes et al., 2010; Tuke et al., 2011).

In fact:

i) all samples were negative for

mitochondrial mouse-specific DNA

sequences;

ii) all the several negative reaction

controls were consistently scored as

negative;

iii) all blank reagent controls were

consistently scored as negative.

These results would exclude the

presence of contaminating mouse or

plasmid DNA in reagents.

In addition, the two positive amplicons

showed different retrovirus sequences,

thus excluding amplicon carry-over

contamination.

The data here reported are relevant to a

small group of CFS patients, and studies

in larger patient cohorts have not

attributed an etiologic role for XMRV or

MLVrelated viruses in CFS (Erlwein et

al., 2010; Switzer et al., 2010; van

Kuppeveld et al., 2010; et al.,

2011).

It is however intriguing that the only

positive results were obtained in these

patients.

On the other hand, in both cases the

positivity could not be confirmed by the

amplification of a different virus gene.

The proviral DNA amount was very low in

our patients, which might explain the

stochastic amplification of a single virus

gene in each of the two positive

patients. Another possibility is that only

fragment of virus DNA might be present

in biologic samples.

In conclusion, while it appears

established that XMRV/MLV sequences

are not detectable in a significant

proportion of CFS patients, the

frequency and the role of evolutionary

relic retrovirus sequences potentially

detectable in the human chromatin

remain to be further elucidated.