FIG. 6.
(A) IF analysis 24 h after transfection of MDBK cells with Bi-NS2ins−, Bi-NS2ins+, and Bi-NS2ins+Δpol RNA (control). No viral antigen was detected in cells transfected with Bi-NS2ins+Δpol RNA. For antibodies see the legend to Fig. 2A. Magnification, ×400. (B and C) GUS release assay with Bi-NS2ins− and Bi-NS2ins+. MDBK cells transfected with replicon RNA were monitored for GUS activity up to 10 days p.t. The GUS activities present in the culture media and adherent cells were determined separately. The total GUS activity of each dish is the sum of the GUS activities detected in the adherent cells and the culture medium. The fraction of the total GUS activity which was detected in the culture medium of a specific dish at a given time point is depicted in the graph as a percentage of the total GUS activity. Values obtained from cultures which originated from the same RNA transfection are indicated by bars with identical patterns. (B) Cell cultures derived from five parallel transfections of Bi-NS2ins− RNA were monitored for up to 10 days at different time points. While at 24 h p.t. one culture of each of the transfections was used for the determination of the GUS activity, three cultures from three independent electroporations were analyzed at 48, 72, and 144 h p.t. At 168 and 240 h p.t., the GUS activities of two cultures originating from two independent electroporations were measured. (C) Three transfections of Bi-NS2ins+ RNA were carried out in parallel, and the cells were monitored for 4 days. Every 24 h one culture of each of the three electroporations was analyzed.