Multiple sources of contamination in samples from patients reported to have XMRV infection

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).

Figure 1. X-SCA amplification profiles of XMRV and MLV templates.

Florescence intensity as a function of cycle number is shown for X-SCA amplifications initiated with dilutions of XMRV RNA and (a) endogenous MLVs found in mouse (TA3.Cyc-T1) genomic DNA or (b) patient plasma samples obtained from WPI and LBS. Red lines: XMRV standards; pink: CFS patient plasma samples; green: Normal control plasma. (c) Aligned sequences of the cloned amplicons detected in (b) from the indicated samples. The XMRV reference sequence matched vp62, virus from 22Rv1 cells, and Pre-XMRV-2.

Figure 2. Neighbor-Joining Phylogenetic Tree of sequences obtained by X-SGS from CFS patients' plasma samples.

Phylogenetic structure of gag single-genome sequences obtained from CFS patients X2 (aqua) X3 (blue) X5 (green), and X8 (red), and TA3.Cyc-T1 mouse cells (black triangles). Also includes sequences of endogenous MLVs extracted from the C57Bl6 genome sequence , as well as XMRV isolates are included for comparison. Where there are multiple identical sequences in the mouse genome, only one is shown, with the number of identical sequences in parentheses.

Figure 3. Env sequences obtained from plasma of patient X8.

(a) Neighbor-joining analysis of full-length env sequences from patient X8, the full length TA3 sequences, and endogenous MLV as shown in Figure 2. (b) Highlighter plot of single-genome sequence alignment of env sequences from patient X8 and from TA3.CycT1 mouse cells.

Figure 4. Env X-SGS of culture supernatants from virus isolations.

(a) Neighbor-joining tree of single-genome env sequences from XMRV infected 293T cell supernatants, 22Rv1 cell supernatants, and supernatants from virus rescue experiments on samples 1–9. (b) Highlighter plots of the same sequences. PreXMRV-1 and -2 and the predicted recombinant (used as outgroup) are included for comparison. The region of multiple crossovers inferred to have occurred between the two parental viruses is shaded.

Figure 5. Gag X-SGS on culture supernatants from isolations.

(a) Neighbor-joining tree of single-genome gag sequences from XMRV infected 293T cell supernatants, 22Rv1 cell supernatants, and supernatants from virus rescue experiments on patients 1–3, and 5. (b) Highlighter plots of the same sequences as compared to XMRV VP62. PreXMRV-1 and -2 and the predicted recombinant (used as outgroup) are included for comparison.