Xenotropic Murine Leukemia Virus-Related Virus (XMRV) is a recently discovered gammaretrovirus reportedly associated with prostate cancer and chronic fatigue syndrome (CFS) [1
]. Urisman et al. first identified XMRV in 2006 in a cohort of prostate cancer patients [2
], followed by Lombardi et al. who reported XMRV infection in 67% of patients with severe CFS and 3.7% of healthy individuals [1
]. These initial reports provided a compelling rationale for further investigations into the prevalence of XMRV infection in human populations. However, controversy arose when subsequent studies failed to detect the virus in similar cohorts [3
]. It was suggested that inconsistencies in detection of XMRV in patient samples could result from varied incidence of infection in different populations, differing criteria for patient selection, and differing detection methods [8
]. It was also proposed that virus levels may be chronically low or episodic in patient plasma or tissues, making virus detection difficult [8
]. Adding to the complexity, detection of XMRV by PCR is highly susceptible to false positive results due to amplification of closely related endogenous Murine Leukemia Viruses (MLVs) in the mouse genome and the high prevalence of contaminating mouse genomic DNA in many specimens and reagents [9
]. Additionally, studies have suggested that XMRV detection is the result of laboratory contamination from infected cell lines [11
]. Paprotka et al. proposed that XMRV originated as a laboratory artifact when two endogenous mouse proviruses recombined during passaging of a human prostate cancer tumor in nude mice, an event that is highly unlikely to have occurred more than once. The authors, therefore, concluded that published XMRV sequences obtained from patient samples must have come from contamination of samples by virus or DNA from cell lines infected with this recombinant virus [14
]. To investigate the human prevalence of XMRV infection, it is clear that reliable detection requires the application of several diagnostic methods used together, including methods that are not influenced by nucleic acid contamination, to avoid reporting potentially high rates of false positives.
Accordingly, we analyzed recently collected blood samples from participants in the MACS cohort using new tests that detect XMRV antibodies and nucleic acid in the blood stream [15
]. The MACS cohort provided the opportunity to assess the association of XMRV with HIV-1 infection and other clinical outcomes and to evaluate its possible mode of transmission. We hypothesized that the prevalence of XMRV infection is higher among men who have acquired HIV-1 infection than among seronegative controls. Previous studies have evaluated samples from HIV-infected cohorts for the presence of XMRV nucleic acid with negative results [7
], but none has looked for the presence of antibody to XMRV.
In the current study, we first screened samples for antibody reactivity to XMRV. This approach eliminated the risk that positive results were due to nucleic acid contamination and mitigated the risk that infection would be missed due to low-level or episodic viremia. To further minimize the risk of reporting false-positive XMRV infection status, we required that antibody and nucleic acid (either viral RNA or DNA) must both be present to report the patient as being XMRV infected. These criteria are supported by studies performed on XMRV-inoculated macaques confirming that both antibody and nucleic acid are readily detectable in longitudinal blood samples collected after XMRV infection (Kearney et al. in press; Del Prete et al. in preparation) [15
]. Plasma samples from the MACS cohort were screened for antibody reactivity by CMIAs and confirmed by Western blot. Reactive samples and blinded, matched antibody negative controls from the same cohort were then tested for the presence of XMRV RNA in plasma and proviral DNA in PBMCs. This approach to determine the prevalence of XMRV infection minimizes the risk of reporting false positives that occur from either cross-reactive antibodies in the bloodstream or contaminating nucleic acid in samples or reagents.