We initially detected very weak signals for XMRV in 15% of patients with CFS with a set of XMRV primers using real-time PCR, but failed to detect XMRV in patients with idiopathic chronic fatigue, chronic inflammatory disorders, other viral infections, or healthy controls. The difference in frequency of a weak PCR positive signal for XMRV for patients with CFS versus controls was not significant, thus, these results did not indicate a clear relationship between XMRV and CFS. Our repeated failure to isolate and clone XMRV sequences by PCR from the samples that were low positive in the real-time PCR assay, suggests that the low positive signals were false-positive artifacts, rather than contamination with mouse DNA. Tuke et al.  reported that Invitrogen Platinum Taq PCR Master Mix was contaminated with mouse DNA sequences; however, they did not detect murine sequences in Applied Biosystems Taq PCR Master Mix. Since we also used Applied Biosystems Taq PCR master mix for amplication of DNA by real-time PCR, this further supports the likelihood of a false positive signal as an artifact, rather than contamination with XMRV. We were also unable to detect XMRV in patients with CFS using a different set of XMRV primers or in PBMCs activated by PHA and further stimulated with IL-2 to amplify XMRV DNA. These latter results are in contrast to those of Lombardi et al. who found that activation of PBMCs from patients with CFS with PHA induced expression of virus proteins and infectious viral particles , which would be expected to amplify XMRV DNA.
Since the original report of Lombardi et al.  in which XMRV DNA was detected in 67% of CFS patients and 3.7% of controls, there are numerous reports from the United Kingdom [3, 4], the Netherlands , China , Japan [12, 23], and the United States [7–10, 19, 24] that did not detect XMRV DNA in patients with CFS. Reasons postulated for the difference in results include geography of the patients, contamination of samples or reagents with mouse DNA, sequence variation in XMRV, and definitions of CFS.
The blood samples we evaluated were collected from the NIH cohort of CFS patients during 1993-1995 and patients were predominantly from Midwestern and Southern United States, but also included patients from Western and Northeastern United States. Lombardi et al. studied patients from the 2006 to 2008, including some patients identified during an outbreak of CFS in 1984-1988, and their patients came from at least 11 states in various regions of the country [1, 17]. Like our cohort, their patients were from diverse areas of the United States. Prior reports generally have studied more recent patients and most of the patients have been from more restricted locations in the United States or different geographic areas than those of the original report. Our study more closely resembles that of Satterfield et al.  who studied patients from multiple states of the continental United States, whereas other studies have reported patients from a single geographic area of the United States [8–11], two states , or other countries [3–6, 23, 25].
Using the nested PCR assay reported by Lo et al. , we detected MLV-related viral DNA in all human samples tested, regardless of whether they were from patients with CFS, inflammatory diseases, or normal controls. Sequence analysis showed that sequences in both CFS and healthy controls aligned with MLV sequences found in mice and reported in patients with CFS or prostate cancer [2, 19, 20]. In contrast, we did not detect MLV-related virus in monkey cell lines. While the nested PCR findings suggest that the human samples were contaminated with mouse DNA, specific testing for mouse genomic DNA (testing for both mouse GAPDH and multi-copy mouse IAP transposons) was negative. In addition, nested PCR of monkey cell DNA did not amplify MLV sequences. Thus, the MLV sequences might be due to contamination during DNA isolation (from the AllPrep DNA/RNA isolation kit), from Platinum Taq (as reported by Tuke et al. , or due to very low levels of MLV related sequences in human DNA. Using other procedures for isolating DNA and PCR, Satterfield et al.  did not detect MLV in CFS patients from 17 states in the United States.
Our serologic analysis by immunoprecipitation of gp70 from XMRV-infected 293T or ferret cells using CFS patient sera and sera from non-CFS control patients did not detect XMRV gp70 specific antibodies in patients with CFS. Our results are consistent with those of Erlwein et al. , Satterfield et al. , Knox et al.  and Shin et al.  who were unable to detect antibody to XMRV in patients with CFS.
Since this work was performed, Paprotka et al  and Knox et al  have shown that XMRV originated sometime between 1993 and 1996 from recombination between two endogenous MLVs during tumor passaging in mice, and that XMRV could not be detected in 43 patients who had previously been reported XMRV positive. Our findings, indicating no definitive evidence linking XMRV with CFS both by PCR and by antibody testing, support those of others that XMRV is not a cause of CFS. Thus, the search must continue for other etiologies for CFS.