Fig. 3.

SBV virions produced in 293-hBST2 are less infectious due to reduced levels of envelope glycoprotein. (A) 293-hBST2 or 293-control cells were infected with SBV (MOI 0.001) and cultured for 48 h when supernatants were collected, filtered through a 0.45 µm filter and the number of SBV genomes quantified by qRT-PCR. CPT-Tert cells were then infected with 2.5 × 10⁵ SBV genome equivalents of these supernatants for one hours at 4 °C to synchronize infection followed by 1.5 h at 37 °C. 8 h post infection cells were fixed and stained by immunofluorescence using an antiserum against SBV N. The number of SBV infected cells was counted (10 fields of view per condition) and compared between groups. Three independent experiments were carried out using three independent viral preparations produced in 293-hBST2 and 293-control cells. Values represent the average of three independent experiments and are presented relative to control cells (100%) (t-test p < 0.05). (B) SBV stocks were produced as described in A and virions pelleted through a 20% sucrose cushion by centrifugation followed by quantitative western blot analysis. The graph displays the average of the Gc/N ratio of at least five independent experiments (t-test p < 0.05). (C) 293-hBST2 or 293-control cells were infected with SBV (MOI 0.001) and total cell lysates were collected at the indicated times post infection and analyzed by quantitative western blotting against SBV N and Gc proteins. The data is expressed relative to the expression of control cells at 22 h post-infection. The graph displays the average of three independent experiments (two-way ANOVA). (D) The area under the curve (AUC) for each of the individual experiments depicted in (C) was estimated and compared between groups (t-test). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.