Type I Interferon Reaction to Viral Infection in Interferon-Competent, Immortalized Cell Lines from the African Fruit Bat Eidolon helvum
Figure 1
Generating immortalized bat cell cultures and measuring interferon (IFN)-β mRNA induction.
(A) Primary bat cell cultures were generated from an embryonic kidney of E. helvum. Cell cultures were immortalized by lentiviral transduction of a simian virus 40 large T antigen. Three clonal cell lines (EidNi/41.1, 41.2 and 41.3) were prepared from a mixed culture (EidNi/41) by end point dilution plating. Bars indicate 20 µm. (B) Phylogenetic relationships of different selected bat species for the generation of target gene sequences (adapted from [3]). For the IFN-β gene four species were selected. (C) Alignment of bat IFN-β genes (E. helvum, M. daubentonii, R. cf. landeri, R. aegyptiacus) and positions of primers and probe for a pan-bat real-time RT-PCR. (D) Replication of a Renilla luciferase expressing Rift valley fever virus clone 13 (RVFV 13) in various cell lines (EidNi/41.3, MEF (mouse embryonic fibroblasts), MA104 (African green monkey kidney) and as reference Vero cells at different MOIs. Cells were infected at different ten-fold diluted MOIs (7.5 until 0.000075) and lysed 24 hpi with Renilla lysis buffer (Promega). Replication was detected by Renilla luciferase read-out. Experiments were performed in duplicates. Highest replication was found in Vero cells followed by EidNi/41.3, MEF, MA104 (between10 to 100-fold less compared to Vero cells). (E) IFN-β mRNA transcription was induced by either RVFV 13 infection (MOI 1) or poly IC transfection (5 µg per 6-well). IFN-β and TATA-box binding protein (housekeeping gene) mRNA was quantified by species-specific real-time RT-PCR assays. The fold induction was calculated with the 2−ΔΔCt method.