Fig. 2.
Generation and characterization of BHK21 cell line stably expressing OHFV-NS1. A Flowchart for cell line selection. VSV-G, m57, and pBABEpuro-OHFV-NS1 plasmids were co-transfected into 293T cells, and obtained retroviral supernatants were filtered with a 0.45-μm filter. BHK21 cells were infected with recombinant retroviral virus containing polybrene. After 24 h, cells were cultured in a medium containing 8 μg/mL puromycin. After several rounds of selection, visible monoclonal cells were picked and propagated. B Selected cell clones were subjected to IFA to determine the expression levels of NS1 using anti-HA antibody. The figure shows 7 cell lines that were 100% IFA positive. C Generation of OHFV-ΔNS1 virus in BHK21NS1 cells. Obtained cell lines were transfected with 1 μg recombinant OHFV-ΔNS1 RNA. Transfected cells were subjected to IFA at 24 and 48 hpt using anti-NS3 antibody.