XMRV Infection in Patients With Prostate Cancer: Novel Serologic Assay and Correlation With PCR and FISH

Detection of XMRV neutralizing antibodies in patient serum and receptor expression of reporter cells. (A, Top) Diagram of single-round reporter gene assay. 293T cells were co-transfected with pSG3Δenv and XRMV env- or HIV env-expression plasmids. Three days later, media was transferred to JC53BL-13 in presence or absence of serially diluted antisera. (A, Bottom) Two days later, cells were lysed and luciferase activity in measured. Relative neutralization (percentage of control) calculated by dividing number of luciferase units at each serum dilution by values in wells containing no test serum and subtracting that value from values in wells containing no test serum. Samples tested in triplicate; error bars represent standard deviation. (B) Detection of XPR1 receptor expression in JC53BL-13 cells. (C) Serum from 20 QQ patients, 10 RQ patients, and 10 RR patients analyzed for neutralizing activity. Horizontal lines represent average percentage of neutralization of samples tested.

PCR and FISH analysis in prostatectomy tissue. (A,B) Two fields of hematoxylin-eosin–stained prostatic tissue and corresponding XMRV FISH positives shown for patient 177. Large panels show FISH positive cells (arrow). Smaller panels show magnified field of hematoxlyin-eosin stain and fluorescent image of XMRV FISH-positive cells. Similar findings were observed for all FISH-positive patients but absent in FISH-negative patients. (C) Representative gel of bands resulting from second round of nested PCR using patient prostatic tissue DNA as template and XMRV-Env gene specific primers. Expected size of band was 217 bp. Each positive PCR product was sequenced to verify XMRV unique sequences. All patient specimens were also negative for mouse mitochondrial DNA, ruling out contamination (data not shown). Sample ID numbers for labeling lanes correspond to sample ID numbers in tables.