Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States

Abstract

Background: XMRV, a xenotropic murine leukemia virus (MuLV)-related virus, was recently identified by PCR testing in 67% of persons with chronic fatigue syndrome (CFS) and in 3.7% of healthy persons from the United States. To investigate the association of XMRV with CFS we tested blood specimens from 51 persons with CFS and 56 healthy persons from the US for evidence of XMRV infection by using serologic and molecular assays. Blinded PCR and serologic testing were performed at the US Centers for Disease Control and Prevention (CDC) and at two additional laboratories.

Results: Archived blood specimens were tested from persons with CFS defined by the 1994 international research case definition and matched healthy controls from Wichita, Kansas and metropolitan, urban, and rural Georgia populations. Serologic testing at CDC utilized a Western blot (WB) assay that showed excellent sensitivity to MuLV and XMRV polyclonal or monoclonal antibodies, and no reactivity on sera from 121 US blood donors or 26 HTLV-and HIV-infected sera. Plasma from 51 CFS cases and plasma from 53 controls were all WB negative. Additional blinded screening of the 51 cases and 53 controls at the Robert Koch Institute using an ELISA employing recombinant Gag and Env XMRV proteins identified weak seroreactivity in one CFS case and a healthy control, which was not confirmed by immunofluorescence. PCR testing at CDC employed a gag and a pol nested PCR assay with a detection threshold of 10 copies in 1 ug of human DNA. DNA specimens from 50 CFS patients and 56 controls and 41 US blood donors were all PCR-negative. Blinded testing by a second nested gag PCR assay at the Blood Systems Research Institute was also negative for DNA specimens from the 50 CFS cases and 56 controls.

Conclusions: We did not find any evidence of infection with XMRV in our U.S. study population of CFS patients or healthy controls by using multiple molecular and serologic assays. These data do not support an association of XMRV with CFS.

Figure 1

Titration of polyclonal MuLV goat antisera in Western blot (WB) assay. Antibody titers of positive control anti-sera and reactivity of pre-immune sera to polytropic MuLV-infected (upper panel) and uninfected (lower panel) HeLa cell crude cell lysates in WB testing. Specific antisera tested are located at the bottom of each WB. Arrows indicate observed titers for each antiserum. Fr, Friend; Ra, Rauscher. Locations of reactivity to specific viral proteins are indicated. Env (gp69/71), envelope; TM (p15E), transmembrane; MA (p15), matrix; Gag (pr68/80); CA (p30), capsid. Molecular weight markers (kD) are provided on the left of the WBs in the upper panels. Sizes of expected viral proteins are provided in each WB in the upper panels.

Figure 2

Titration of polyclonal XMRV rabbit and monoclonal spleen focus forming virus (SFFV) envelope rat antisera in Western blot (WB) assay. Antibody titers of positive control anti-sera and reactivity of pre-immune sera to polytropic MuLV-infected (upper panel) and uninfected (lower panel) HeLa cell crude cell lysates in WB testing. Specific antisera tested are located at the bottom of each WB. Arrows indicate observed titers for each antiserum. Fr, Friend; Ra, Rauscher. Locations of reactivity to specific viral proteins are indicated. Env (gp69/71), envelope; TM (p15E), transmembrane; MA (p15), matrix; Gag (pr68/80); CA (p30), capsid. Molecular weight markers (kD) are provided on the left of the WBs in the upper panels. Sizes of expected viral proteins are provided in each WB in the upper panels.

Figure 3

Absence of XMRV antibodies in CFS patients by Western blot (WB) analysis. Representative WB results for CFS cases from Wichita and Georgia identified after unblinding. Determination of MuLV specific reactivity is determined by comparison of observed seroreactivity to polytropic MuLV-infected HeLa antigens and uninfected HeLa antigens in upper and lower panels, respectively. Lanes 1 - 4 and 5 - 8 are plasma from CFS cases from the population based studies in Georgia and Wichita, respectively; lanes 9 - 12 are physician-referred CFS cases from the Georgia Registry study. MuLV positive and negative goat serum controls are labelled.

Figure 4

Absence of XMRV antibodies in CFS patients and healthy persons by ELISA using recombinant XMRV proteins. Representative XMRV Envelope (Env) ELISA results for 50 CFS cases and 49 healthy persons identified after unblinding. Specimens coded with W and G1-G50 are from the population-based study in Wichita and Georgia, respectively; specimens G59 - G75 are from physician-referred CFS cases from the Georgia Registry study. Specimens from a healthy control and a person with CFS, coded as G6 and G9 respectively, were weakly seroreactive in this test but were not confirmed by either Western blot or immunofluorscence testing. Human sera were diluted 1:200. The human negative control serum was obtained from a healthy volunteer previously determined to be seronegative. The polyclonal mouse Env antiserum was diluted 1:100. Assay cut-off was determined by the mean of the test samples plus three standard deviations.

Figure 5

Absence of XMRV polymerase (pol) sequences in CFS patients. A. Representative nested pol PCR results using PBMC DNA specimens from persons with CFS identified after unblinding. Lanes 1 - 5, 6 - 10, and 11 - 14 are results for persons with CFS from Wichita, Georgia, and the Georgia registry studies, respectively; lanes 15 and 16, water only controls; lane 17, negative human PBMC DNA control; lanes 18 and 19, assay sensitivity controls consisting of 10¹ and 10³ copies of XMRV VP62 plasmid DNA diluted in a background of 1 ug of human PBMC DNA, respectively. B. Semi-quantitative β-actin PCR results for PBMC DNA specimens above in lanes 1 - 14; lane 15, water control; lanes 16 - 19, 10-fold dilutions of blood donor PBMC DNA starting at 0.1 ug as a positive assay control.

Figure 6

Absence of XMRV gag sequences in CFS patients. A. Representative nested gag PCR results from patients from the Georgia Registry identified after unblinding. Lanes 1 and 20, 100-bp ladder; lanes 2 - 15 are results from CFS patients; lanes 16 - 18 assay sensitivity controls consisting of 10, 3 and 1 copies of XMRV VP62 plasmid DNA diluted in a background of 250 ng of human PBMC DNA; lane 19, water control. B. GAPDH PCR results for same PBMC DNA specimens above.