Figure 1.
Failure of detection of XMRV nucleic acids in plasma and whole blood of CFS patients and healthy controls.
A) First round PCR products of a representative number of RNA samples isolated from patient plasma using primers 419F and 1154R. A 10−5 dilution of 22Rv1 cell culture supernatant and three known concentrations of XMRV plasmid DNA were included as controls. B) Second round amplification products of nested PCR using primers Gag-I-F and Gag-I-R of samples shown in A). Identical results were obtained with primers NP116 and NP117 (see text, data not shown). The detection limit was below 1 copy/ µl isolated RNA or 5 copies/reaction. C) Results of qRT-PCR for XMRV plasmid control in serial dilutions ranging from 106 to 102 copies/ml as well as negative controls for both primer pairs used, F2/R2 (upper panel) and WPI (lower panel). All patient plasma and whole blood samples were found to be negative after a total of 45 amplification cycles (data not shown).
Figure 2.
No evidence for infectious virus or XMRV-specific antibodies in plasma of CFS patients and healthy controls.
A) GFP expression of DERSE.Li-G cells 7 days (upper panels) or 21 days (lower panels) after spinoculation with two different dilutions of 22Rv1 cell culture supernatants (10−4 and 10−6 dilution) or patient plasma. No GFP expression could be observed in any of the cells inoculated with human plasma. B) Immunoblotting of C7-purified XMRV antigen with patient plasma for detection of anti-XMRV/MLV antibodies. Representative WB results for CFS patients and healthy controls. Lane 1, anti-Friend MuLV whole virus, goat polyclonal antisera; lane 2, anti-Rauscher MuLV envelope, goat polyclonal antisera; lane 3, XMRV negative blood donor plasma. Locations of reactivity to specific viral proteins are indicated; Env (gp69/71), envelope; TM (p15E), transmembrane; MA (p15), matrix; Gag (pr68); CA (p30), capsid.