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No Evidence of Murine-Like Gammaretroviruses in CFS Patients Previously Identified as XMRV-Infected

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Science  01 Jul 2011:
Vol. 333, Issue 6038, pp. 94-97
DOI: 10.1126/science.1204963
  • Fig. 1

    (A) Representative nested gag PCR results using genomic DNA (gDNA) from P1 patient leukocytes. A negative control (water, lane 0) and two positive controls [XMRV gag plasmid at 10 and 100 copies per reaction (rxn)] were included in each run. As control, patient DNA was also tested with single-round PCR for RNaseL (17). DNA markers (M) and the positions of expected PCR products are annotated. (B) Representative nested RT-PCR results on P2 PBMC samples. Positive and negative controls are shown. Ten-fold serial dilutions of XMRV gag plasmid control start at 1000 copies per reaction. Negative controls for each reaction step were tested in triplicate: *RNA/DNA extraction negative control, **RT control, and ***PCR control.

  • Fig. 2

    Evaluation of 60 CFS plasma samples for the presence of XMRV antibodies. (A) Two recombinant protein–based CMIAs were used to detect specific antibodies to XMRV gp70 and p15E proteins (17). The x axis represents the CMIA signal in a unit of natural log–transformed ratio of sample signal to the cutoff signal (log N of S/CO). (B) Western blot analysis of gp70 CMIA reactive CFS sample using native XMRV viral proteins and mammalian-expressed recombinant gp70 protein. Sample keys: the gp70 CMIA-reactive (CFS) sample 09-7571, positive control (PC) of XMRV-infected macaque plasma, negative control (NC) of normal blood donor, and molecular mass (MW) markers in kilodaltons (kD).

  • Fig. 3

    Effects of human serum on xenotropic MLV and XMRV. Shown is the percent serum inactivation of virus, as measured by induction of focus formation in mink S+L– cells by control untreated X-MLV and XMRV (17). Representative results are shown. Unheated sera from 12 other CFS patients gave similar findings with nearly complete inactivation of X-MLV and partial to high inactivation of XMRV. The X-MLV was obtained from New Zealand Black (NZB) mouse cells and propagated in mink lung cells (20). XMRV was obtained from the human prostate cell line (22Rv1). For the five studies conducted, the control virus titers measured as focus formation in mink S+L– cells were 126, 430, 168, 246, and 208 foci (X-MLV); 84, 376, 208, 284, and 206 foci (XMRV). N, control; P, CFS patient (see table S1); black bars, X-MLV unheated sera; shaded bars, X-MLV heated sera; white bars, XMRV unheated sera; hatched bars, XMRV heated sera.

  • Table 1

    Summary of assays used to evaluate blood samples from CFS patients in P2. Information about the CFS patients is provided in table S1. Two subjects were studied twice within a 3-month period (table S1) and gave the same results.

    AssayPercent XMRV-positive
    (n)
    PCR analysis of PBMC-derived DNA0 (0/31)
    RT-PCR analysis of patient plasma0 (0/31)
    PBMC culture fluids*0 (0/19)
    Reverse transcriptase assay of supernatants from mink lung cells passaged after PBMC coculture*0 (0/30)†

    *Infectious virus assay: Fluids were tested for infectious virus production by reverse transcriptase and the mink S+L– cell assays (see text) (17).

    †Insufficient cells were available for these studies from subject no. 24.

    Additional Files

    • No Evidence of Murine-Like Gammaretroviruses in CFS Patients Previously Identified as XMRV-Infected

      Konstance Knox, Donald Carrigan, Graham Simmons, Fernando Teque, Yanchen Zhou, John Hackett Jr., Xiaoxing Qiu, Ka-Cheung Luk, Gerald Schochetman, Allyn Knox, Andreas M. Kogelnik, and Jay A. Levy

      Materials/Methods, Supporting Text, Tables, Figures, and/or References

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      • Materials and Methods
      • Figs. S1 to S3
      • Tables S1 to S3
      • References

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