Fig 4.

wt BTV-1 and BTV-1/NS3mCherry are able to replicate in D. melanogaster. (A) Specific PCR detection of W. pipientis before and after tetracycline treatment. Before tetracycline treatment, the Yolk-Gal4 strain (Yolk-NT) was found to be positive for W. pipientis using primers specific for 16S rRNA genes and wsp. In contrast, and as expected, the tetracycline-treated Yolk-Gal4 (Yolk-T) and Canton-S (CS) strains were found negative for W. pipientis. PCR amplification of mitochondrial 12S rRNA gene primers was used as a DNA extraction control. (B) Yolk-Gal4 D. melanogaster flies were injected with BTV-1/NS3mCherry and either harvested immediately (day 0, input virus) or incubated for 10 days prior to harvest (day 10). Each individual was homogenized and used to infect KC cells. Five days p.i., cells were analyzed by FACS. (C) Tetracycline-treated (Yolk-T) and untreated (Yolk-NT) D. melanogaster flies were injected with BTV-1/NS3mCherry (BTV) or virus-free medium (mock) and assayed as described above. D. melanogaster harvested immediately after injection (D0) showed very weak evidence of infection compared to those which had been mock injected (left versus middle panel). After 10 days of incubation (D10), the Yolk-T consistently showed high levels of infection, whereas the Yolk-NT showed only minimal evidence of viral replication (right panels). (D) The graph displays the percentage of mCherry-positive KC cells inoculated with D. melanogaster homogenates and harvested 5 days postinoculation. Horizontal bars represent the mean values of the data obtained from three independent experiments (5 individuals per day and per experiment). No significant difference was observed between day 0 and day 10 Yolk-NT homogenates (P = 0.156), whereas Yolk-T homogenates were highly permissive to BTV-1/NS3mCherry infection (P = 4 × 10⁻⁶). (E and F) Yolk-T (E) and Canton-S (F) D. melanogaster females were injected intrathoracically with wt BTV-1 or BTV-1/NS3mCherry at the same viral titer (2 × 10⁴ PFU/ml) and either harvested immediately (day 0, input virus) or incubated for 10 days prior to collection (day 10). Each individual was homogenized and titrated by dilution assays on BSR cells. Horizontal bars represent the mean values of the data obtained (10 individuals per day and per experiment). All P values indicated between D0 and D10 titers are significant (P < 10⁻⁴).