Using highly sensitive PCRs with primers that detect XMRV and primers that detect MuLV-like sequences, no proviral DNA was detected in any of the prostate cancer samples independently of murine DNA contamination. This served to confirm our previous studies in which FFPE prostate tissue was tested and XMRV/MuLV sequences failed to be amplified [28
]. Here we have added further data to show that no XMRV or MuLV-like sequences can be detected in fresh UK prostate tissue or in prostate cancer samples collected from Japan. Samples from India showed evidence of MuLV and XMRV sequences when viral genomic sequences were amplified by nested PCR. However, this was concordant with murine genomic DNA contamination detected using primers to IAP. IAPs are retrotransposons present at the level of around 1000 copies per mouse genome [30
]. Thus, IAP PCR represents a highly sensitive detection method for murine DNA. Although the sample size was small (n
= 10), we found no evidence to suggest that XMRV might be involved in other cancers, such as diffuse large B-cell lymphoma.
It was reported last year that XMRV had been detected in greater than 60% of 50 samples from English blood donors [36
]. In contrast, we found no evidence of XMRV or pMuLV in any of 540 whole-blood samples from unselected NHSBT donors nor were we able to detect MuLV-like sequences in either the DNA from whole blood or cDNA prepared from the plasma minipools from donors in England. There are three possible explanations for this. Firstly, there are no MuLV infections in blood donors in England. Secondly, there are MuLV infections, but that the assays used failed to detect them, either due to sensitivity or sequence variation. Thirdly, there are MuLV infections, but the prevalence is too low to be detected in the sample sizes tested.
Research into the presence of MuLVs in the human population is contentious, given discrepant findings [37
]. Contamination from sequences contained in apparently XMRV-positive samples, amplified products, or plasmids has been suggested as a reason for the finding of MuLVs in human samples [30
]. A study of XMRV in patients with CFS or chronic immunomodulatory conditions, using Invitrogen Platinum Taq (IPT), reported a gag
sequence with >99% homology to a mouse endogenous retrovirus [19
]. This was designated as contamination, although the paper failed to speculate on the source of this sequence. Sato and colleagues (2010) recently reported finding predominantly RNA sequences, related to a pMuLV, in IPT containing reagents [30
]. Another study concluded that the detection of MuLV-related sequences in human samples could be due to contamination with mouse DNA, most likely contained in various laboratory reagents [29
]. We have demonstrated that murine sequences can be present in prostate sections, resulting in false positive detection of XMRV [28
]. A phylogenetic overview concluded that the proviral sequences present in the genome of 22Rv1 cell line were ancestral to the published XMRV sequences [31
]; finally, it has been shown that the mapping of integration sites of XMRV in prostate cancer tissues, thought to unequivocally confirm the existence of XMRV in clinical samples, was at least partially contaminant derived [41
], further emphasising the ease with which contamination can occur.
The sources of contamination are still to be fully elucidated. However, given that most retroviral laboratories have worked with MuLV or MuLV-derived vector systems, or at least used murine reagents, it is essential that sufficient appropriate controls are included in all PCRs.
The absence of MuLVs from all the samples analysed in this study, where there was no concomitant detection of murine genomic sequences, adds weight to the growing body of data questioning the evidence for murine retrovirus infection of humans [42
]. It is always challenging to prove a negative result, but it is likely that XMRV will be added to the long list of RNA rumour viruses [43