Multiple Sources of Contamination in Samples from Patients Reported to Have XMRV Infection

Francis W Ruscetti,Wei Shao,Elizabeth M Anderson,Mary F Kearney,Steve H Hughes,Ann Wiegand,Frank Maldarelli,John W Mellors,John M Coffin,Stuart F J Le Grice,Jonathan Spindler

doi:10.1371/journal.pone.0030889

Abstract

Xenotropic murine leukemia virus (MLV)-related retrovirus (XMRV) was reported to be associated with prostate cancer by Urisman, et al. in 2006 and chronic fatigue syndrome (CFS) by Lombardi, et al. in 2009. To investigate this association, we independently evaluated plasma samples from 4 patients with CFS reported by Lombardi, et al. to have XMRV infection and from 5 healthy controls reported to be XMRV uninfected. We also analyzed viral sequences obtained from supernatants of cell cultures found to contain XMRV after coculture with 9 clinical samples from 8 patients. A qPCR assay capable of distinguishing XMRV from endogenous MLVs showed that the viral sequences detected in the CFS patient plasma behaved like endogenous MLVs and not XMRV. Single-genome sequences (N = 89) from CFS patient plasma were indistinguishable from endogenous MLVs found in the mouse genome that are distinct from XMRV. By contrast, XMRV sequences were detected by qPCR in 2 of the 5 plasma samples from healthy controls (sequencing of the qPCR product confirmed XMRV not MLV). Single-genome sequences (N = 234) from the 9 culture supernatants reportedly positive for XMRV were indistinguishable from XMRV sequences obtained from 22Rv1 and XMRV-contaminated 293T cell-lines. These results indicate that MLV DNA detected in the plasma samples from CFS patients evaluated in this study was from contaminating mouse genomic DNA and that XMRV detected in plasma samples from healthy controls and in cultures of patient samples was due to cross-contamination with XMRV (virus or nucleic acid).

Highlights

In 2006, a novel infectious agent, xenotropic murine leukemia virus (MLV)-related virus (XMRV), was identified by hybridization to an oligonucleotide chip (‘‘virochip’’) and reported to be associated with prostate cancer [1]
These results show that the retroviral sequences detected in the chronic fatigue syndrome (CFS) plasma samples were more similar to endogenous MLV DNA than xenotropic MLV-related virus (XMRV) RNA, suggesting high levels of contamination with mouse genomic DNA
These findings, taken together with the fact that XMRV originated by recombination between two endogenous MLVs in a xenograft of prostate cancer passaged in nude mice, makes it likely that sample contamination with XMRV was from cloned or PCR-amplified XMRV DNA

Summary

Introduction

In 2006, a novel infectious agent, xenotropic MLV-related virus (XMRV), was identified by hybridization to an oligonucleotide chip (‘‘virochip’’) and reported to be associated with prostate cancer [1]. XMRV infection was reported to be associated with chronic fatigue syndrome (CFS) by Lombardi, et al who detected XMRV, using PCR, in 67% of samples from CFS patients compared to 3.7% of samples from healthy controls [2] Such high frequencies of XMRV infection prompted concerns about widespread XMRV infection and stimulated research to determine the prevalence of XMRV infection worldwide. Strong evidence that XMRV detection was the result of laboratory contamination came from a recent report that XMRV originated as a recombinant virus between two endogenous MLV proviruses (PreXMRV-1 and PreXMRV2) between 1993 and 1996 during passage of a human prostate cancer xenograft in nude mice [18]. Derived from cancers passaged in nude mice, have been shown to be infected with viruses derived from a variety of endogenous MLVs; these viruses are distinct from XMRV [15,20,21]

Methods

Results

Conclusion