Quite fascinating how the story of XMRV and prostate cancer is so similar to XMRV and CFS but I figured we should stick to the part that seeks to explain the contradictions seen in both PS and CFS studies i.e. why perhaps some studies were ‘positive’ and others not:
‘By 2010, the XMRV literature was filled with stark, inescapable contradictions and concerns were being raised regarding false-positive results in assays for XMRV caused by laboratory-based contamination.
Detection of XMRV and related viruses in clinical samples often relied on PCR for detection of viral nucleic acids; however, mice contain endogenous viral sequences that can be amplified with “XMRV-specific” primers in PCR-based assays.40
Thus, PCR assays for XMRV are particularly susceptible to false-positive results owing to the presence of the MLV genomes in mouse DNA and the extraordinary sensitivity of the assays. Every mouse cell contains at least 100 MLV genomes.
Therefore, a millionth of a microliter of mouse blood contains sequences that can potentially be ampli¬fied in “XMRV-specific” PCR. As mice are ubiquitous in biomedical research, it can be very difficult to obtain clinical samples without trace amounts of murine DNA.
An important step that helped to clarify the role of contaminating mouse DNA in XMRV assays was the development of PCR assays for murine sequences, including mitochon¬drial cytochrome oxidase and intracisternal A particle (IAP) sequences.41
As there are approximately 1,000 copies of the IAP retrotransposon in the mouse genome, testing for IAP sequences is an extremely sensitive way of detecting the presence of mouse DNA. When these assays were applied to a series of clinical samples, every sample that had been scored as XMRV-positive was also found to contain IAP sequences.41,42
A number of commercial reagents used in assays for XMRV (such as Taq polymerases, PCR master mixes, RT PCR kits and extraction columns) have also been found to contain trace amounts of murine nucleic acids and might, therefore, explain positive results.43–45
In some studies, XMRV is detected more frequently in samples from patients with a disease than control subjects. This appar¬ent association of the virus with disease is, at first glance, difficult to reconcile with the idea that the positive results represent contamination.
However, it must be con¬sidered that the disease samples might be collected or handled differently, or at dif¬ferent times, compared with the controls.
As suggested by Weiss,2 it is also possible that disease samples might simply be handled more often than “job-lot” control samples, increasing the opportunities for potential contamination to occur.
Given the extraordinary sensitivity of PCR, which can detect single molecules of template, it seems likely that it could even detect airborne nucleic acid molecules in the laboratory.
A very important development in recon¬ciling the contradictions between different laboratories has emerged from a joint study organized as the Blood XMRV Scientific Research Working Group (SRWG).46
This group sent blinded samples (positive control samples spiked with XMRV, nega¬tive control samples with no XMRV, and clinical samples from patients previously reported to be positive for XMRV) to nine different laboratories for testing by nested PCR, virus culture and serology.
Two laboratories that had collaborated in the original report on XMRV in CFS samples36 were the only laboratories to report detection of XMRV in clinical samples.
However, these two laboratories also “detected” XMRV in negative control samples that did not contain the virus, providing direct, unequivocal evidence that the assays used in these laboratories suffer from some artifacts producing positive results in the absence of XMRV.46
Of particular note, in the SRWG study, the Lo et al.39 labora¬tory found no XMRV in any of five of their previously reported positive samples or in 10 samples previously reported as positive by Lombardi et al.,36 despite their ability to detect XMRV in all five of the spiked positive controls.46
IHC has also been used to detect XMRV in clinical samples. Surprisingly, this method seemed more sensitive than PCR in the work of Schlaberg et al.8
One concern regarding this work is the way in which the anti-XMRV antiserum was generated whole viral particles produced in human cells were used as the immunogen.
However, retrovirus particles contain cellular proteins in addition to viral proteins.47,48 Thus, this immunization is likely to have generated antibodies against human proteins, as well as against the viral proteins.
Stieler et al.22 have reported that the antiserum used by Schlaberg can recognize cellular proteins in non-infected human and mouse cell lines. Furthermore, Sakuma et al.16 compared the staining by an anti-MLV p30/gp70 antibody that can detect XMRV precursor Gag, CA, and Env proteins to that by the anti-XMRV antiserum used by Schlaberg and colleagues8 in IHC assays on prostate cancer tissues.
Both antibodies reproducibly stained 293T cells transfected with an XMRV clone in positive control assays. However, only the anti-XMRV antiserum used by Schlaberg et al.8 stained areas of prostate tumor epithelium, whereas the anti-MLV p30/gp70 antibody did not show positive staining in any of the tissues.
Sakuma et al.16 concluded that “we cannot detect XMRV in prostate cancer tissues and that the antibody described by Schlaberg, Singh and colleagues recognizes non-viral proteins in addition to XMRV”.16
In our own studies, we received prostate cancer tissue sections (kindly provided by Dr Ila Singh, University of Utah) from a number of the same patients tested by Schlaberg et al.8 The samples were predicted to be XMRV-positive based on the previous IHC results with the anti-XMRV antiserum.8
However, the sections did not stain with two different broadly reactive MLV antisera that had previously detected XMRV in positive control assays.18 Taken together, the evidence suggests that it is highly unlikely that the IHC staining observed by Schlaberg et al.8 represents the true presence of XMRV.
Finally, one surprising feature of all XMRV sequences reported in clinical samples is their uniformity. Five sequences purportedly isolated from patient samples were found to be, on average, >99.9% identical to each other at the nucleotide level.49
This near-identity of XMRV sequences is difficult to reconcile with the error-prone nature of retroviral replication. In fact, different HIV 1 genomes isolated from a single infected individual show far more divergence than is seen in the entire set of XMRV sequences published to date.
However, it should be noted that the sequence diversity of different isolates of human T cell leukemia virus type 1 (HTLV) (a member of the deltaretrovirus genus, distinct from both MLVs and lentiretroviruses such as HIV 1) is also far lower than that of HIV 1, although not as low as that in XMRV.50
As sequence diversity is generated during the viral replication cycle, it is likely that there is much less ongoing viral replication in an HTLV-infected individual than in an individual infected with HIV 1.51 The level of replication in an MLV-infected, viremic mouse is not known.'
