XMRV -Comments in Retrovirology nr.2

[Guest guest]

Reference:

*Comment XMRV -Mouse DNA

contamination -Retrovirology*

Help ME Circle, 22 January 2011

See Co-Cure: http://bit.ly/ibnHjS

~jvr

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RETROVIROLOGY

Short report

An Endogenous Murine Leukemia Viral

Genome Contaminant in a Commercial

RT-PCR Kit is Amplified Using Standard

Primers for XMRV

Eiji Sato1, Rika A Furuta2 and Takayuki Miyazawa1

1 Laboratory of Signal Transduction, Institute for

Virus Research, Kyoto University, 53

Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507,

Japan

2 Japanese Red Cross Osaka Blood Center, 2-4-43

Morinomiya, Joto-ku, Osaka 536-8505, Japan

Retrovirology 2010, 7:110

doi:10.1186/1742-4690-7-110

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This report *An Endogenous Murine Leukemia Viral

Genome Contaminant in a Commercial RT-PCR Kit is

Amplified Using Standard Primers for XMRV* by Sato

E. et al, can be found at: http://bit.ly/gJrkSn

~jvr

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http://bit.ly/gEPneT

Comments

Sato et al missed a trick

Isabel Greenwood

(12 January 2011)

PA institute

A number of retrovirologists have expressed a belief

that the recent discovery of MLV related viruses in

people with Myalgic Encephalomyelitis also called

Chronic Fatigue Syndrome (ME/CFS) and Prostate

Cancer Patients are in fact contaminants.

Four studies were posted simultaneously in this

journal in Dec 2010 where the authors offered their

interpretation of their findings as evidence in support

of their hypothesis.

Others have argued that alternative explanations of

their findings are readily available.

They argue that even without other lines of

evidence, not considered in these studies, an

alternative interpretation argues against the

advocates of contamination argument.

This letter focuses on a critique of one of these

papers namely the one authored by Sato and others

(1).

The abstract is included for the readers perusal.

*....During pilot studies to investigate the presence

of viral RNA of xenotropic murine leukemia virus

(MLV)-related virus (XMRV) infection in sera from

chronic fatigue syndrome (CFS) patients in Japan, a

positive band was frequently detected at the

expected product size in negative control samples

when detecting a partial gag region of XMRV using a

one-step RT-PCR kit.

We suspected that the kit itself might have been

contaminated with small traces of endogenous MLV

genome or XMRV and attempted to evaluate the

quality of the kit in two independent laboratories.

We purchased four one-step RT-PCR kits from

Invitrogen, TaKaRa, Promega and QIAGEN in Japan.

To amplify the partial gag gene of XMRV or other

MLV-related viruses, primer sets (419F and 1154R,

and GAG-I-F and GAG-I-R) which have been widely

used in XMRV studies were employed.

The nucleotide sequences of the amplicons were

determined and compared with deposited sequences

of a polytropic endogenous MLV (PmERV), XMRV and

endogenous MLV-related viruses derived from CFS

patients.

We found that the enzyme mixtures of the one-step

RT-PCR kit from Invitrogen were contaminated with

RNA derived from PmERV.

The nucleotide sequence of a partial gag region of

the contaminant amplified by RT-PCR was nearly

identical (99.4 % identity) to a PmERV on

chromosome 7 and highly similar (96.9 to 97.6 %) to

recently identified MLV-like viruses derived from CFS

patients.

We also determined the nucleotide sequence of a

partial env region of the contaminant and found that

it was almost identical (99.6 %) to the PmERV.

In the investigation of XMRV infection in patients of

CFS and prostate cancer, researchers should

prudently evaluate the test kits for the presence of

endogenous MLV as well as XMRV genomes prior to

PCR and RT-PCR tests....*

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This analysis will now focus on two extracts from the

abstract which form the foundation of the entire

study.

*....We purchased four one-step RT-PCR kits from

Invitrogen, TaKaRa, Promega and QIAGEN in Japan.

To amplify the partial gag gene of XMRV or other

MLV-related viruses, primer sets (419F and 1154R,

and GAG-I-F and GAG-I-R) which have been widely

used in XMRV studies were employed....*

Sato et al point to the fact that these primer sets

have been widely used in XMRV studies.

They do not state however that these primer sets

used in a one step RT-PCR or a single round PCR

have never been able to detect even population

levels of XMRV (3, 4) or any other MLV related

viruses.

Indeed son et al (5) demonstrated that primer

sets which could readily detect XMRV sequences in

vitro could not do so in a patient sample known to

be infected, by a PCR assay targeting the env gene,

or serological methods.

Thus these primers in studies could not have

detected this RNA sequence because they could

not detect a MULV sequence of any kind.

*....We also determined the nucleotide sequence of

a partial env region of the contaminant and found

that it was almost identical (99.6 %) to the

PmERV....*

Sato and others relate that they partially sequenced

the env region. One must ask why they did not

sequence the entire env region which would then

have enabled the virus to be identified.

This is most unfortunate. In fact no region of the

virus which would have enabled unequivical

identification of the virus was sequenced at all.

The PCR assays used by Sato could not have

detected a contaminant in any XMRV study because

when they were used no MLV or MLV related virus of

any kind was detected.

The authors here have not taken any of the available

opportunities to identify the virus. It is therefore

difficult so see why this study has been presented as

offering evidence in support of those who argue

contamination when it clearly does no such thing.

1) Eiji Sato, Rika A Furuta, Takayuki Miyazawa; An

endogenous murine leukemia viral genome

contaminant in a commercial RT-PCR Kit is amplified

using standard primers for XMRV; Retrovirology 2010,

7:110 (20 Dec. 2010)

2) HC Groom, VC Boucherit, K Makinson, E Randal, S

Baptista, S Hagean JW Gow, FM Mattes, JR Kerr, JP

Stoye and KN Boshop; Absence of xenotropic murine

leukaemia virus-related virus in UK patients with

chronic fatigue syndrome; Retrovirology 2010,

7:10doi:10.1186/1742-4690-7-10

3) FJM van Kuppeveld, AS de Jong, KH Lanke, GW

Verhaegh, WJG Melchers, CMA Swanink, G

Bleijenberg, MG Netea, JMD Galama, & JWM van der

Meer (2010); Prevalence of xenotropic murine

leukaemia virus-related virus in patients with chronic

fatigue syndrome in the Netherlands: retrospective

analysis of samples from an established cohort;

British Medical Journal : 10.1136/bmj.c1018

4) BP son, GE Ayala and JT Kimata; Detection

of Xenotropic Murine Leukemia Virus-Related Virus in

Normal and Tumor Tissue of Patients from the

Southern United States with Prostate Cancer Is

Dependent on Specific Polymerase Chain Reaction

Conditions; J Infect Dis. (2010) 202 (10): 1470-1477.

doi: 10.1086/656146

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lombardi et al advised

not to use one step kits

Gerwyn

(21 January 2011)

PA institute

If the Sato and others had read the original study by

Lombardi et al (2009) they would have noted that

the authors of that study advised (in the text)

against the use of one step kits because mouse

contamination had been detected in such kits.

It seems strange that Sato and others have

attemped to *reinvent the wheel* in their study by

presenting information that was already in the public

domain