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Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome

Chronic fatigue syndrome (CFS) is a debilitating disease of unknown etiology that is estimated to affect 17 million people worldwide. Studying peripheral blood mononuclear cells (PBMCs) from CFS patients, we identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus–related virus (XMRV), in 68 of 101 patients (67%) as compared to 8 of 218 (3.7%) healthy controls. Cell culture experiments revealed that patient-derived XMRV is infectious and that both cell-associated and cell-free transmission of the virus are possible. Secondary viral infections were established in uninfected primary lymphocytes and indicator cell lines after their exposure to activated PBMCs, B cells, T cells, or plasma derived from CFS patients. These findings raise the possibility that XMRV may be a contributing factor in the pathogenesis of CFS.

Sequences of full-length XMRV genomes from two CFS patients and a partial genome from a third patient were generated (table S1). CFS XMRV strains 1106 and 1178 each differed by 6 nt from the reference prostate cancer strain XMRV VP62 (EF185282 [GenBank] ), and with the exception of 1 nt, the variant nucleotides mapped to different locations within the XMRV genome, suggesting independent infections. In comparison, prostate cancer–derived XMRV strains VP35 and VP42 differed from VP62 by 13 and 10 nt, respectively. Thus, the complete XMRV genomes in these CFS patients were >99% identical in sequence to those detected in patients with prostate cancer. To exclude the possibility that we were detecting a murine leukemia virus (MLV) laboratory contaminant, we determined the phylogenetic relationship among endogenous (non-ecotropic) MLV sequences, XMRV sequences, and sequences from CFS patients 1104, 1106, and 1178 (fig. S2). XMRV sequences from the CFS patients clustered with the XMRV sequences from prostate cancer cases and formed a branch distinct from non-ecotropic MLVs common in inbred mouse strains. Thus, the virus detected in the CFS patients’ blood samples is unlikely to be a contaminant.

To determine whether XMRV proteins were expressed in PBMCs from CFS patients, we developed intracellular flow cytometry (IFC) and Western blot assays, using antibodies (Abs) with novel viralspecificities. These antibodies included, among others, (i) rat monoclonal antibody (mAb) to the spleen focus-forming virus (SFFV) envelope (Env), which reacts with all polytropic and xenotropic MLVs (7); (ii) goat antisera to whole mouse NZB xenotropic MLV; and (iii) a rat mAb to MLV p30 Gag (8). All of these Abs detected the human VP62 XMRV strain grown in human Raji, LNCaP, and Sup-T1 cells (fig. S3) (5). IFC of activated lymphocytes (6, 9) revealed that 19 of 30 PBMC samples from CFS patients reacted with the mAb to MLV p30 Gag (Fig. 2A). The majority of the 19 positive samples also reacted with antisera to other purified MLV proteins (fig. S4A). In contrast, 16 healthy control PBMC cultures tested negative (Fig. 2A and fig. S4A). These results were confirmed by Western blots (Fig. 2, B and C) (6) using Abs to SFFV Env, mouse xenotropic MLV, and MLV p30 Gag. Samples from five healthy donors exhibited no expression of XMRV proteins (Fig. 2C). The frequencies of CFS cases versus healthy controls that were positive and negative for XMRV sequences were used to calculate a Pearson {chi}2 value of 154 (two-tailed P value of 8.1 x 10–35). These data yield an odds ratio of 54.1 (a 95% confidence interval of 23.8 to 122), suggesting a nonrandom association with XMRV and CFS patients.

To determine which types of lymphocytes in blood express XMRV, we isolated B and T cells from one patient’s PBMCs (6). Using mAb to MLV p30 Gag and IFC, we found that both activated T and B cells were infected with XMRV (Fig. 2D and fig. S4A). Furthermore, using mAb to SFFV Env, we found that >95% of the cells in a B cell line developed from another patient were positive for XMRV Env (fig. S4B). XMRV protein expression in CFS patient–derived activated T and B cells grown for 42 days in culture was confirmed by Western blots (fig. S4C) using Abs to SFFV Env and xenotropic MLV.

We next investigated whether the viral proteins detected in PBMCs from CFS patients represent infectious XMRV. Activated lymphocytes (6) were cocultured with LNCaP, a prostate cancer cell line with defects in both the JAK-STAT and RNase L pathways (10, 11) that was previously shown to be permissive for XMRV infection (12). After coculture with activated PBMCs from CFS patients, LNCaP cells expressed XMRV Env and multiple XMRV Gag proteins when analyzed by Western blot (Fig. 3A) and IFC (fig. S5A). Transmission electron microscopy (EM) of the infected LNCaP cells (Fig. 3B), as well as virus preparations from these cells (Fig. 3C), revealed 90- to 100-nm-diameter budding particles consistent with a gamma (type C) retrovirus (13).

We also found that XMRV could be transmitted from CFS patient plasma to LNCaP cells when we applied a virus centrifugation protocol to enhance infectivity (6, 14, 15). Both XMRV gp70 Env and p30 Gag were abundantly expressed in LNCaP cells incubated with plasma samples from 10 of 12 CFS patients, whereas no viral protein expression was detected in LNCaP cells incubated with plasma samples from 12 healthy donors (Fig. 4A). Likewise, LNCaP cells incubated with patient plasma tested positive for XMRV p30 Gag in IFC assays (fig. S5B). We also observed cell-free transmission of XMRV from the PBMCs of CFS patients to the T cell line SupT1 (Fig. 4B) and both primary and secondary transmission of cell-free virus from the activated T cells of CFS patients to normal T cell cultures (Fig. 4C). Together, these results suggest that both cell-associated and cell-free transmission of CFS-associated XMRV are possible.

We next investigated whether XMRV stimulates an immune response in CFS patients. For this purpose, we developed a flow cytometry assay that allowed us to detect Abs to XMRV Env by exploiting its close homology to SFFV Env (16). Plasma from 9 out of 18 CFS patients infected with XMRV reacted with a mouse B cell line expressing recombinant SFFV Env (BaF3ER-SFFV-Env) but not to SFFV Env negative control cells (BaF3ER), analogous to the binding of the SFFV Env mAb to these cells (Fig. 4D and S6A). In contrast, plasma from seven healthy donors did not react (Fig. 4D and fig. S6A). Furthermore, all nine positive plasma samples from CFS patients but none of the plasma samples from healthy donors blocked the binding of the SFFV Env mAb to SFFV Env on the cell surface (fig. S6B). These results are consistent with the hypothesis that CFS patients mount a specific immune response to XMRV.Neurological maladies and immune dysfunction with inflammatory cytokine and chemokine up-regulation are some of the most commonly reported features associated with CFS. Several retroviruses,including the MLVs and the primate retroviruses HIV and HTLV-1, are associated with neurological diseases as well as cancer (17). Studies of retrovirus-induced neurodegeneration in rodent models have indicated that vascular and inflammatory changes mediated by cytokines and chemokines precede the neurological pathology (18, 19). The presence of infectious XMRV in lymphocytes may account for some of these observations of altered immune responsiveness and neurological function in CFS patients.

We have discovered a highly significant association between the XMRV retrovirus and CFS. This observation raises several important questions. Is XMRV infection a causal factor in the pathogenesis of CFS or a passenger virus in the immunosuppressed CFS patient population? What is the relationship between XMRV infection status and the presence or absence of other viruses that are often associated with CFS (e.g., herpesviruses)? Conceivably these viruses could be cofactors in pathogenesis, as is the case for HIV-mediated disease, in which co-infecting pathogens play an important role (20). Patients with CFS have an elevated incidence of cancer (21). Does XMRV infection alter the risk of cancer development in CFS? As noted above, XMRV has been detected in prostate tumors from patients expressing a specific genetic variant of the RNASEL gene (5). In contrast, in our study of this CFS cohort, we found that XMRV infection status does not correlate with the RNASEL genotype (6) (table S2).

Finally, it is worth noting that 3.7% of the healthy donors in our study tested positive for XMRV sequences. This suggests that several million Americans may be infected with a retrovirus of as yet unknown pathogenic potential.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1179052/DC1

Materials and Methods

Figs. S1 to S6

Tables S1 and S2

References

References and Notes

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  • 22. We thank D. Bertolette, Y. Huang, C. Hanson, and J. Troxler for technical assistance; K. Nagashima for EM; and C. Ware and K. Hunter for discussions. Funded by the Whittemore Peterson Institute and the Whittemore Family Foundation; the National Cancer Institute (NCI); NIH (under contract HHSN26120080001E); and grants to R.H.S. from NCI/NIH (CA104943), the U.S. DoD Prostate Cancer Research Program (W81XWH-07-1338), the V Foundation for Cancer Research, the Charlotte Geyer Foundation, and Mal and Lea Bank. The content of this publication does not reflect the views or policies of the U.S. DHHS, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. R.H.S. may receive royalty payments in the future from Abbott Laboratories. GenBank accession numbers are as follows: WPI-1130, GQ483508 [GenBank] ; WPI-1138, GQ483509 [GenBank] ; WPI-1169, GQ483510 [GenBank] ; WPI-1178, GQ497343 [GenBank] ; WPI-1106, GQ497344 [GenBank] ; and WPI-1104, GQ497345.Note added in proof: V.C.L. is operations manager of Viral Immune Pathologies Laboratory, which is in negotiations with the Whittemore Peterson Institute to offer a dianostic test for XMRV.
Received for publication 14 July 2009. Accepted for publication 31 August 2009.

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