[Note: according to Whittemore Peterson Institute, this Japanese study “used WPI techniques of PBMC culture and serology for positive detection of XMRV in blood samples.”]
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
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: Reported by Whittemore Peterson, posted on DiagnoseSupport.com. Furuta RA, Miyazawa T, Sugiyama T, Kimura T, Hirayama F, Tani Y, Shibata H. Department of Research, Japanese Red Cross Osaka Blood Center; Laboratory for Viral pathogenesis, Institute for Virus Research, Kyoto University; Department of Urology, Nishiwaki Municipal Hospital.