Fig. 1.

Replication of XMRV in CEM and CEM-SS cells. (A) Schematic overview of the experimental protocol. Supernatant containing XMRV was harvested from 22Rv1 cells, and XMRV RNA copies in the supernatant were determined by quantitative real-time RT-PCR using primers and a probe for a region of XMRV env. Culture supernatants containing different amounts of XMRV RNA copies were then used to infect 1 × 10⁶ CEM or CEM-SS cells. At 6 h postinfection, cells were extensively washed, and culture supernatants were harvested every day from days 0 to 14. XMRV RNA copy numbers were determined by quantitative real-time RT-PCR. (B to G) Replication kinetics of XMRV 22Rvl in CEM and CEM-SS cells infected with 22Rv1 culture supernatant containing 10⁴ XMRV RNA copies (B), 10⁵ XMRV RNA copies (C), 10⁶ XMRV RNA copies (D), 10⁷ XMRV RNA copies (E), 10⁸ XMRV RNA copies (F), or 10¹⁰ XMRV RNA copies (G). The viral RNA in the supernatant of infected cells was isolated, and XMRV RNA was detected by quantitative real-time RT-PCR. The results are plotted as copies of viral XMRV RNA per 8.4 μl of culture supernatant of infected cells. The detection limit for quantitative RT-PCR was 1.1 × 10² copies/8.4 μl (indicated as a solid line). A representative experiment from two independent experiments is shown. Error bars indicate standard deviations. (H) Determination of increases in viral RNA copies in CEM and CEM-SS cells 14 days postinfection. The increase in viral RNA copies produced from CEM and CEM-SS cells infected with different amounts of 22Rvl XMRV are shown in panels B to G. The average number of RNA copies per 8.4 μl for days 12 to 14 was divided by the average number of RNA copies per 8.4 μl from days 1 to 3.