Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome

Abstract

Background: Detection of a retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), has recently been reported in 67% of patients with chronic fatigue syndrome. We have studied a total of 170 samples from chronic fatigue syndrome patients from two UK cohorts and 395 controls for evidence of XMRV infection by looking either for the presence of viral nucleic acids using quantitative PCR (limit of detection <16 viral copies) or for the presence of serological responses using a virus neutralisation assay.

Results: We have not identified XMRV DNA in any samples by PCR (0/299). Some serum samples showed XMRV neutralising activity (26/565) but only one of these positive sera came from a CFS patient. Most of the positive sera were also able to neutralise MLV particles pseudotyped with envelope proteins from other viruses, including vesicular stomatitis virus, indicating significant cross-reactivity in serological responses. Four positive samples were specific for XMRV.

Conclusions: No association between XMRV infection and CFS was observed in the samples tested, either by PCR or serological methodologies. The non-specific neutralisation observed in multiple serum samples suggests that it is unlikely that these responses were elicited by XMRV and highlights the danger of over-estimating XMRV frequency based on serological assays. In spite of this, we believe that the detection of neutralising activity that did not inhibit VSV-G pseudotyped MLV in at least four human serum samples indicates that XMRV infection may occur in the general population, although with currently uncertain outcomes.

Figure 1

Sensitivity of PCR screening for XMRV in PBMC DNA. VP62 plasmid was serially diluted 1:10 into PBMC DNA from a healthy donor and tested by Taqman PCR with env 6173 primers and probe. The final amount of VP62 DNA in the reaction was A, 2.3 × 10^-2 ng, B, 2.3 × 10^-3 ng, C, 2.3 × 10^-4 ng, D, 2.3 × 10^-5 ng, E, 2.3 × 10^-6 ng, F, 2.3 × 10^-7 ng or G, 2.3 × 10^-8 ng. The limit of sensitivity was 2.3 × 10^-7 ng (shown by trace F) which equates to 16 molecules of VP62 XMRV clone.

Figure 2

Examples of BLT positive serum neutralisation activity. A, Infectivity of XMRV (measured as counts per second of chemiluminescence produced from β-galactosidase activity) after incubation with patient serum or hybridoma cell supernatant. Infectivity is plotted against the reciprocal dilution of the BLT serum (black circles, top panel, sample Q488, bottom panel, sample Q610; triangles, negative control, monoclonal 603; squares, positive control, monoclonal 83A25'). The dashed line indicates viral infectivity in the absence of sera. B, Infectivity data for viruses with four different envelopes (circles, XMRV; squares, MLV(X); triangles, Friend-MLV; crosses, VSV-G) after incubation with patient serum. Data were normalised by setting the infectivity for each virus in the absence of patient serum at 100%, and plotted against the reciprocal of serum dilution for two positive sera, top panel sample Q488 and bottom panel sample Q610.

Figure 3

Distribution of neutralisation activity in three samples sets. Numbers of patients showing different degrees (>70% , 50-70% and <50%) of neutralisation of XMRV infectivity are shown for the 1/40 and 1/80 serum dilutions. A, Total BLT cohort (n = 226); B, SGUL CFS cohort (n = 142); C, SGUL control blood donor cohort (n = 157).

Figure 4

Examples of SGUL positive serum neutralisation activity. A, Infectivity of XMRV (measured as counts per second of chemiluminescence produced from β-galactosidase activity) after incubation with patient serum or hybridoma cell supernatant. Infectivity is plotted against the reciprocal dilution of the SGUL serum (black circles, top panel, sample Q321, bottom panel, sample Q323; triangles, negative control, monoclonal 613; squares, positive control, monoclonal 83A25'). The dashed line indicates viral infectivity in the absence of sera. B, Infectivity data for viruses with four different envelopes (circles, XMRV; squares, MLV(X); triangles, Friend-MLV; crosses, VSV-G) after incubation with patient serum. Data were normalised by setting the infectivity for each virus in the absence of patient serum at 100%, and plotted against the reciprocal of serum dilution for two positive sera, top panel sample Q321 and bottom panel sample Q323.