Serologic and PCR testing of persons with chronic fatigue syndrome in the United States shows no association with xenotropic or polytropic murine leukemia virus-related viruses

Abstract

In 2009, a newly discovered human retrovirus, xenotropic murine leukemia virus (MuLV)-related virus (XMRV), was reported by Lombardi et al. in 67% of persons from the US with chronic fatigue syndrome (CFS) by PCR detection of gag sequences. Although six subsequent studies have been negative for XMRV, CFS was defined more broadly using only the CDC or Oxford criteria and samples from the US were limited in geographic diversity, both potentially reducing the chances of identifying XMRV positive CFS cases. A seventh study recently found polytropic MuLV sequences, but not XMRV, in a high proportion of persons with CFS. Here we tested blood specimens from 45 CFS cases and 42 persons without CFS from over 20 states in the United States for both XMRV and MuLV. The CFS patients all had a minimum of 6 months of post-exertional malaise and a high degree of disability, the same key symptoms described in the Lombardi et al. study. Using highly sensitive and generic DNA and RNA PCR tests, and a new Western blot assay employing purified whole XMRV as antigen, we found no evidence of XMRV or MuLV in all 45 CFS cases and in the 42 persons without CFS. Our findings, together with previous negative reports, do not suggest an association of XMRV or MuLV in the majority of CFS cases.

Figure 1

Absence of antibodies to XMRV in plasma from persons with and without CFS from the US. a. Antibody titers of positive control anti-sera to purified XMRV antigen in WB testing. Specific antisera tested are provided at the top of each WB. Arrows indicate observed titers for each antiserum. Locations of reactivity to specific viral proteins are indicated. Env (gp69/71), envelope; TM (p15E), transmembrane; MA (p15), matrix; Gag (pr68); CA (p30), capsid. Molecular weight markers (kD) are provided on the right of the WB. Sizes of expected viral proteins are provided to the left of the WB. b. Detection of XMRV antibodies in three experimentally-infected macaques (RII, RYh and RLq). Days post infection and immunization with XMRV are shown with arrows [16]. Locations of reactivity to specific viral proteins are indicated. Env (gp69/71), envelope; TM (p15E), transmembrane; MA (p15), matrix; Gag (pr68); CA (p30), capsid. Molecular weight markers (kD) are provided on the right of the WB. Sizes of expected viral proteins are provided to the left of the WB. c. Representative WB results for CFS cases and persons without CFS. Lane 1, 1:250 dilution of anti-Friend MuLV whole virus, goat polyclonal antisera; lane 2, XMRV negative blood donor plasma; lanes 3, 4, 9, 11 are plasma from persons without CFS; lanes 5 - 8, 10, 12, 14 - 17, 19, 20, 23 - 27 are plasma from persons with severe CFS; lanes 13, 18, 21, and 22 are plasma from persons with unclassified CFS. Locations of reactivity to specific viral proteins are indicated; Env (gp69/71), envelope; TM (p15E), transmembrane; MA (p15), matrix; Gag (pr68); CA (p30), capsid. Molecular weight markers (kD) are provided on the left of the WB.

Figure 2

Absence of XMRV/MuLV sequences by PCR of PBMC DNA of persons with and without CFS from the US. Representative nested polymerase (pol2) PCR results. Lanes 1 and 2 are results from persons without CFS; lanes 3 - 8, 10 and 11 are results from patients classified with severe CFS; lanes 9, and 12 - 14 are results from patients with unclassified CFS; lane 15, negative human PBMC DNA control; lanes 16 and 17, water only controls; lanes 18 and 19, assay sensitivity controls consisting of 10¹ and 10³ copies of XMRV VP62 plasmid DNA diluted in a background of 1 μg of human PBMC DNA, respectively.

Figure 3

Absence of XMRV/MuLV sequences by real-time PCR in PBMC DNA of persons with and without CFS from the US. Representative real-time XMRV polymerase (pol) PCR results. Upper panel; pol amplification plot using XMRV synthetic DNA diluted in a background of 2.5 ug of DNA from whole blood to 12,000, 1,200, 120 and 12 copies and negative (water and DNA) controls demonstrating the sensitivity and dynamic linear range of the assay. Lower panel; pol amplification plot for DNA from 40 persons, including 18 with severe CFS, 8 with unclassified CFS, and 14 without CFS. Two positive controls (DNA from 17 XMRV infected 22Rv1 cells spiked into 2.5 μg of human leukocyte DNA) for the pol PCR, and two negative controls (2.5 μg of DNA) are also shown. Only the two positive controls were detected in this testing.