Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors

Abstract

Chronic fatigue syndrome (CFS) is a serious systemic illness of unknown cause. A recent study identified DNA from a xenotropic murine leukemia virus-related virus (XMRV) in peripheral blood mononuclear cells (PBMCs) from 68 of 101 patients (67%) by nested PCR, as compared with 8 of 218 (3.7%) healthy controls. However, four subsequent reports failed to detect any murine leukemia virus (MLV)-related virus gene sequences in blood of CFS patients. We examined 41 PBMC-derived DNA samples from 37 patients meeting accepted diagnostic criteria for CFS and found MLV-like virus gag gene sequences in 32 of 37 (86.5%) compared with only 3 of 44 (6.8%) healthy volunteer blood donors. No evidence of mouse DNA contamination was detected in the PCR assay system or the clinical samples. Seven of 8 gag-positive patients tested again positive in a sample obtained nearly 15 y later. In contrast to the reported findings of near-genetic identity of all XMRVs, we identified a genetically diverse group of MLV-related viruses. The gag and env sequences from CFS patients were more closely related to those of polytropic mouse endogenous retroviruses than to those of XMRVs and were even less closely related to those of ecotropic MLVs. Further studies are needed to determine whether the same strong association with MLV-related viruses is found in other groups of patients with CFS, whether these viruses play a causative role in the development of CFS, and whether they represent a threat to the blood supply.

Fig. 1.

MLV-related gag gene sequences detected in blood DNA from CFS patients. (A) Results of PBMC DNA from CFS patient samples 1–25 (of 41 samples examined) amplified after the first round of nested PCR using a previously published primer set (419F/1154R): targeting gag gene. (B) Results of PBMC DNA from the 25 CFS samples after completing the second round of nested PCR using an in-house–designed PCR primer set (NP116/NP117). (C) MLV-related gag gene RNA sequences are detected in plasma of CFS samples by RT-nested PCR. Results of RT-nested PCR for RNA derived from the plasma samples of CFS patients 4–17 are shown. The positions of expected sizes of the “positive” PCR amplicons are indicated by arrows. M, DNA ladder size markers. All positive PCR amplicons with the expected size have been confirmed by DNA sequencing.

Fig. 2.

MLV-related gag gene sequences detected in normal blood donors by nested PCR. (A) Results of PBMC DNA from blood donors 1–25 (of 44 donors examined) amplified after the first round of nested PCR using primer set 419F/1154R. Lane 4: PBMC DNA from BD22 has a positive target PCR amplicon confirmed by sequencing. (B) Results of PBMC DNA from the 25 normal blood donors after the second round of nested PCR using PCR primer set GAG-I-F/GAG-I-R (4). Sequencing of the PCR bands with size ∼413 bp revealed that lane 4 (BD22), lane 7 (BD26), and lane 9 (BD28) were MLV-like virus gag gene sequences; lane 8 (BD27) was a human sequence. The positions of expected sizes of the positive PCR amplicons are indicated by arrows. M, DNA ladder size markers.

Fig. 3.

Phylogenetic trees corresponding to the MSAs shown in Figs. S1 and S2 were generated by the ClustalW2 program using the neighbor-joining method (Materials and Methods). (A) Phylogenetic analysis based on the 746-nt gag gene nucleotide sequences amplified from blood samples of CFS patients and BD-22 of the corresponding MSA in Fig. S1. (B) Phylogenetic analysis based on the 380-nt gag gene sequences amplified from blood samples of CFS patients and healthy blood donors using the primer set NP116/NP117 of the corresponding MSA in Fig. S2.

Fig. 4.

Phylogenetic analysis of protein sequences based on the alignment shown in Fig. S3. CFS types 1, 2, and 3 and BD-22 and MLVs gag protein sequences are compared. Gag protein sequences starting from the AUG initiation codon are aligned with those of relevant endogenous as well as exogenous MLVs. Sequences of MLVs are referred to as polytropic (P), ecotropic (E), amphotropic (A), or modified polytropic (Mpmv). MoMLV, Moloney murine leukemia virus; HEMV, hortulanus endogenous murine virus.

Fig. 5.

Comparison of sensitivity in amplifying mouse DNA by the semi-nested PCR targeting mouse-specific mtDNA and by the nested PCR targeting MLV-like virus gag gene. Serial dilutions of mouse spleen DNA (from 40 pg to 2.5 fg) were spiked into 35 ng of total human PBMC DNA and compared in parallel for the mouse DNA detection sensitivity of the two PCR assays. (A) In the first round of the mtDNA-specific PCR assay, 10 fg or more of mouse DNA could be detected in the presence of 35 ng of human DNA by producing the 286-bp target product. In the first round of MLV gag gene nested PCR assay, 10 pg or more of mouse DNA could be detected in the presence of 35 ng of human DNA by producing the ∼730-bp target product. (B) In the second round of mouse-specific mtDNA semi-nested PCR, the 153-bp target amplicon could consistently be amplified from 2.5 fg of mouse DNA. In the second round of gag gene-specific nested PCR, the 413-bp target product could be amplified from 0.5 pg or more of mouse DNA. Lane 0 fg: 35 ng of human DNA without spiking any mouse DNA. Lane H₂O: No DNA template. M: 100-bp DNA ladder mix. Primers and PCR cycle numbers used in each round of amplification for both of the assays are shown at the top of each gel.

Fig. 6.

Testing of CFS patients’ and healthy blood donors’ samples positive for MLV-like gag gene sequences for the presence of mouse DNA contamination using the semi-nested PCR assay targeting mouse-specific mtDNA. Serial dilutions of mouse DNA were spiked into 35 ng of human DNA and used as the controls of the assay sensitivity. The first round of mouse mtDNA semi-nested PCR (A) detected 10 fg of mouse DNA, and the second round of the semi-nested PCR (B) detected 2.5 fg of mouse DNA in the presence of 35 ng human background DNA. No evidence of mouse DNA contamination could be found by either round of mouse mtDNA semi-nested PCR in the PBMC DNA (35 ng) of CFS patients (patients 8, 17, 20, and 25); three blood donors (BD-22, BD-26, BD-28) tested positive and two blood donors (BD-21 and BD-23) tested negative for the MLV-like virus gag gene sequences. Healthy blood donors’ samples positive for MLV-like gag gene sequences are labeled by asterisks. Lane 0: 35 ng of human DNA without spiking any mouse DNA. Lane H₂O: No DNA template. M: 100-bp DNA ladder mix.