Japanese study detects XMRV using Whittemore Peterson Institute techniques

Saw a link to this today on the WPI's website. http://www.wpinstitute.org/news/news_current.html

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THE PREVALENCE OF XMRV IN HEALTHY BLOOD DONORS IN JAPAN

XMRV Research


THE PREVALENCE OF XENOTROPIC MURINE LEUKEMIA VIRUS-RELATED VIRUS IN
HEALTHY BLOOD DONORS IN JAPAN

Rika A. Furuta1, Takayuki Miyazawa2, Takeki Sugiyama3, Takafumi
Kimura1, Fumiya Hirayama1, Yoshihiko Tani1 and Hirotoshi Shibata1

1 Department of Research, Japanese Red Cross Osaka Blood Center, 2
Laboratory for Viral pathogenesis, Institute for Virus Research, Kyoto
University, 3 Department of Urology, Nishiwaki Municipal Hospital.


To estimate the impacts of infection with xenotropic murine leukemia
virus-related virus (XMRV) on the blood service, we investigated the
prevalence of this virus in both prostate cancer patients and healthy
blood donors in Japan. All specimens from the prostate cancer patients
were collected after obtaining their written informed consent. The
ethical committee of the Japanese Red Cross Society approved the
examination of XMRV antibodies, but not nucleic acids, in random donor
sera.

All serum samples of healthy blood donors tested negative for HIV-1,
HIV-2, HTLV-1, hepatitis B virus, hepatitis C virus and human
parvovirus B19.

To make a recombinant virus as test antigens for the antibody
screening, 293T cells were transfected with an expression vector
carrying an XMRV provirus clone, namely, VP62 (kindly gifted by Dr. R.
H. Silverman). We used an env-defective mutant of HIV-1 derived from
pNL4-3 (kindly gifted by Dr. A. Adachi) as a negative control.

Two days after transfection, the culture supernatants of the
transfected cells were collected and concentrated 20 times by
centrifugation. We implemented western blotting assay to screen
antibodies against XMRV in sera because a high background was observed
if we performed enzyme-linked immunosorbent assay. In the western
blotting, the blot strips were incubated with the serum samples
diluted 1:100 with 5 % skim milk in Tris-buffered saline overnight at
4C.

Two of 32 serum samples collected from the prostate cancer patients
and 5 of 300 serum samples collected from healthy blood donors tested
positive for antibodies against XMRV Gag protein. We did not observe
any specific signals against Env proteins in the western blotting. Of
the 2 serum samples that were obtained from prostate cancer patient
and tested positive for anti-XMRV antibodies, the XMRV specific
nucleic acid sequence was detected in only one sample (patient #24) by
using nested RT-PCR.

In addition, we collected 7 mL of whole blood cells from the patient
#24 and cultured the peripheral blood mononuclear cells (PBMCs) in the
presence of recombinant interleukin 2 and concanavalin A.

The PBMCs were harvested after 10 days of culture, the virus was
isolated from the cells by performing a LacZ marker rescue assay and
RNA and genomic DNA were extracted. The nested PCR performed to detect
XMRV yielded positive results for both genomic and RT PCR, although
the virus was successfully isolated in only 1 of 3 independent
experiments. To examine the susceptibility of PBMCs derived from
healthy individuals to XMRV, we inoculated activated PBMCs from 3
healthy volunteers with the culture supernatant of the PBMCs obtained
from patient #24. By using the nested PCR, we detected the
XMRV-specific nucleic acid sequence in the genomic DNA of the PBMCs
obtained from 2 of the 3 healthy volunteers.

We conclude that XMRV infection is prevalent among both prostate
cancer patients and healthy individuals in Japan. Although our study
had a limited sample size, the prevalence among blood donors as
determined by identifying XMRV-specific antibodies was found to be
1.7%, while that among prostate cancer patients was found to be 6.3%
(P<0.05, one-sided Mann-Whitney U-test).

The results of genomic PCR performing on the PBMCs indicate that XMRV
is sustained in a few fractions of blood cells and can spread through
blood even though the virus replication rate appears to be very low.


Source: 1 Department of Research, Japanese Red Cross Osaka Blood Center, 2
Laboratory for Viral pathogenesis, Institute for Virus Research, Kyoto
University, 3 Department of Urology, Nishiwaki Municipal Hospital.

This is good news then!!

Excellent. They should look at cfs patients next.

I saw this too but am a bit confused...the link that the WPI provides is
http://www.diagnosesupport.com/heal...ted-virus-in&catid=132:xmrv-research&Itemid=8

The date on that post is December 31,2009. So is this new info or did we already know this?

Has this been properly published anywhere yet?

I was hoping this would be a CFS study...

Esther, I agree, but I guess the exciting thing about this is that they were able to replicate WPI's findings using their methods. Where the other studies to date have not even attempted to replicate them and were therefore unable to find XMRV.

This is a Japanese study that was never published, but presented at a conference and cited in XMRV papers. It is still not a full paper published in a peer-reviewed journal, but just the abstract on a website. What is new is that the WPI has linked to it and said that the Japanese researchers used WPI methods.

This has been discussed on this thread: http://www.forums.aboutmecfs.org/showthread.php?3238-Who-found-XMRV-in-Japan

Exactly!! This study suggests faulty technique with the other (so called) replication studies. It also also suggests to me that faulty technique was more the problem than cohort/selection, or even geographics.

WPI shouldn't have put this as breaking news although it might be new? to them. This was the Japanese study presented 5/2009 that was discussed on the forum recently and for which a member (ukxmrv) e-mailed Cold Spring Harbor and got the abstract. The part about it using WPI methods is also unclear to me. It's similar in that the Japanese team used a variety of methods (PCR, culture, Western Blot, antibody) to find XMRV but I don't know if the exact methods are those used by the WPI.

This is not a peer-reviewed study so I fail to see how it matches up with the four published XMRV CFS studies, let alone illustrate a problem with the failed validation attempts. In fact isn't this just the Japanese study that has been talked about for months? Hardly 'breaking news' except that it now appears on a new XMRV website.

Unfortunately, there is not enough in this abstract to tell if they really did use the WPI method. Given that this is actually a new report of an older study by the Japanese Red Cross, I seriously doubt they had contact with WPI. But perhaps they used the same type of culture as WPI, that might be worth reporting. Or is there some undisclosed relationship between this group and WPI?

Still, they may have really found 1.7% XMRV positives in blood donors. And the problems in the study do support the notion that PCR testing does not always work right for XMRV, it appears their PCR could not confirm all the antibody results, something WPI has been saying now for awhile. Interesting....

This thread really ought to be combined with this one:

Who found XMRV in Japan?

This is the first reference I found to this study and it was from the first UK paper. This has been discussed previously in the other thread.

pcr does not detect latent virus full stop. Have a look at the Plos one peer review criterea when you have the time. See what you think of the process and the time it takes.

The criterea for publishing make intersting reading too .Why did the BMJ break their own peer review rules when publishing the dutch study/

I didn't know about the Japanese study. Didn't register in my foggy brain when I read the first UK study.

The fact they found xmrv in healthy controls reinforces for me just how arrogant and out of touch McClure, Wessely, the Dutch study people and BMJ are- trying to trash WPI when the evidence points to the conclusion that they are the ones who are probably not doing the science well enough to pick up xmrv cases.