Additionally, the failure to detect XMRV may be attributable to differences in the detection techniques employed. We have found that detection of XMRV required rather specific conditions. For instance, at least 600 ng of prostate tissue DNA was necessary for reliable detection with our PCR assay. XMRV was detected in 3.2% of the patients when we initially used 100–140 ng of prostate tissue DNA, compared with 22.2% of the patients when we used 650 ng. Additionally, we found that detection of XMRV from patient specimens, but not from LNCaP cells infected in vitro, depended on the gene targeted in the PCR assay. We were unable to detect XMRV in the patient tissue samples by nested PCR with primers specific for the gag and pol genes, regardless of whether 100 or 650 ng of DNA was used as template. We found the gag primers to be at least 10-fold less sensitive than the env primers, and the pol primers tended to amplify a competing region from the human genome (data not shown). It is unclear whether these deficiencies account for the inability to detect XMRV in patient samples or whether XMRV is mainly present as an incomplete provirus in the cells of these patients. Nonetheless, the difficulty associated with detecting XMRV in patient samples may perhaps explain studies that do not detect the virus among large cohorts. We found our nested PCR assay for XMRV env to be capable of detecting 1 infected cell per 1105 uninfected LNCaP cells in 1 of 3 samples with use of 600 ng of DNA. The fact that the PCR-positive tissue specimens tested positive in only 1 or 2 of 3 replicates may indicate that XMRV provirus is present at a very low copy number.
In conclusion, our data support a hypothesis that XMRV is endemic to North America.