FIG. 8.

Packaging of pol I-driven minigenome segments into CCHF particles. Murine (pRF243 and pRF284) and human (pRF249 and pRF283) pol I-driven CCHF virus vRNA and cRNA minigenomes (Fig. 1C) were transfected into BHK-21 or 293T cells 24 h prior to superinfection with CCHF virus (MOI, 0.001 PFU/cell). Cell lysates were prepared 48 h later and assayed for CAT activity, while the cell culture media were collected and 2-ml undiluted samples were transferred to fresh HuH-7 or VeroE6 cells (P1). This was repeated twice (P2 and P3), and CAT activity was assayed after each passage. (A) vRNA minigenomes. Two different cell lines (HuH-7 and VeroE6) were used for passaging recombinant CCHF viruses derived from either the murine (lanes 3 to 8) or human (lanes 11 to 16) pol I system. Lane 1, pRF243-transfected but nonsuperinfected BHK-21 cells; lane 2, pRF243-transfected and superinfected BHK-21 cells; lanes 3 to 5, P1 to P3 in HuH-7 cells; lanes 6 to 8, P1 to P3 in VeroE6 cells; lane 9, pRF249-transfected but nonsuperinfected 293T cells; lane 10, pRF249-transfected and superinfected 293T cells; lanes 11 to 13, P1 to P3 in HuH-7 cells; lanes 14 to 16, P1 to P3 in VeroE6 cells. (B) cRNA minigenomes. VeroE6 cells were used for passaging recombinant CCHF viruses derived from either the human (lanes 2 to 5) or murine (lane 7 to 10) pol I system. Lane 1, pRF283-transfected but nonsuperinfected 293T cells; lane 2, pRF283-transfected and superinfected 293T cells; lanes 3 to 5, P1 to P3 in VeroE6 cells; lane 6, pRF284-transfected but nonsuperinfected BHK-21 cells; lane 7, pRF284-transfected and superinfected BHK-21 cells; lanes 8 to 10, P1 to P3 in VeroE6 cells. The results of two independent experiments are summarized.