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The Prevalence Of Xmlv In Blood Donors In Japan


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#1 akela

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Inviato 16 marzo 2010 - 22:31:48

XMRV Research
Thursday, 31 December 2009 15:20

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
4░C.

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.


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Messaggio modificato da akela, 16 marzo 2010 - 22:35:13





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