Figure 3
Generation of an hACE2-transgenic bat cell line for infection experiments with ORF8 variant rSCV. (a) Picture of an African Rhinolophus alcyone bat (copyright Victor Max Corman). Primary cell culture and immortalization of R. alcyone embryonic lung cells (b) was done as described in the Material and Methods section. To determine the RhiLu cell type the epithelial protein marker cytokeratin (c) and the fibroblast marker S-100A4 (d) were detected by immunofluorescence assay using mouse-anti cytokeratin or rabbit-anti S-100A4 Ig. Secondary detection was performed by incubating with goat-anti-mouse cyanin 2- or goat-anti-rabbit cyanin 3-labeled Igs. The bars represent 20 µm. (e) Integration of hACE2 into the genome of RhiLu cells was verified by PCR. The vector containing the hACE2-puromycin resistance gene construct was used as a PCR positive control. (f) Expression of hACE2 was confirmed by Western blot analysis using mouse anti-hACE2 Ig (1:1,000). In addition, β actin protein was detected using rabbit anti-β-actin Ig (1:2,000) to ensure that similar protein amounts were applied. MA104 cells, expressing hACE2 naturally, served as positive control. (g) Virus replication of rSCV8full, rSCVepi and rSCVdel8 was observed by titration of supernatants sampled at 24, 48, and 72 hpi. Cells were infected in triplicates at an MOI of 0.001. Error bars represent standard deviation of the mean.
