Fig 2: Production of infectious NSDV progeny in SRD-12B can be restored by SKI-1/S1P complementation. Non-confluent SRD-12B cells were transfected with pcDNA3.1 plasmid encoding human SKI-1/S1P. Transfected cells were infected 8 h post transfection with NSDV at a MOI of 0.1 and cultivated for up to 72 h post infection (p.i.). Samples were collected at the indicated times p.i. for subsequent analyses. Three independent experiments were performed in duplicates. (A) Detection of SKI-1/S1P, viral NP and ß-actin expression in transiently transfected SRD-12B cells by western blot analysis. Cell lysates were collected, proteins separated by SDS-PAGE and target proteins detected using commercial MBTPS1 antibodies (Invitrogen), rabbit-derived anti-NP antibodies and anti-ß-actin antibodies. Labeled proteins were visualized by incubation with HRP-labeled secondary antibodies. One representative blot from three independent experiments is shown. (B) Replication of NSDV in CHO-K1, SRD-12B and S1P-complemented SRD-12B cells (S1P_SRD-12B). Supernatants were harvested and titrated by TCID50. The growth curves shown display the mean titers plus SEM of three independent experiments. (C) Viral S segment RNA levels in the cell supernatant. The amount of viral S segment RNA in the supernatant was determined by RT-qPCR and expressed as mean copy numbers per microliter. Error bars depict the standard error of the mean (SEM). (D) Infectivity of virus progeny from cell supernatant of CHO-K1, SRD-12B and S1P_SRD-12B cells was determined by transfer to highly susceptible SW13 cells. Supernatants from NSDV-infected CHO-K1, SRD-12B and S1P_SRD-12B cells were collected at 24 h p.i. and transferred to confluent SW13 cells. Inoculum was removed after 1 h and wells were intensively washed before incubation for 24 h. SW13 cells were then fixed before polyclonal anti-NP antibodies were added. Labeled proteins were visualized using Alexa Fluor 488-conjugated secondary antibodies. Nuclei were counterstained with Hoechst 33,342. Representative images from two independent experiments are shown. Magnification, 20x. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)