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. 2018 May 1;8:127. doi: 10.3389/fcimb.2018.00127

Figure 4.

Figure 4

The Tet-On 3G NS1-expressing Vero cell line facilitates the propagation of the NS1 R38A/K41A virus. The rescued WT and NS1 R38A/K41A virus of F1 were blindly passaged in Vero cells. The same volume was used in all infections. Virus titers (A) of F2 to F5 WT and NS1 R38A/K41A virus in Vero cells were measured in MDCK cells by plaque assays. The 293T cells were transfected with 0, 4, or 8 μg of FLAG-NS1 plasmid and then infected with NS1 R38A/K41A virus at MOI of 0.01. At 48 h p.t., the cells were lysed for western blot analysis (B). NS1 and M1 were detected with anti-FLAG or M1 antibodies. β-actin was probed as the loading control. Tet-On 3G NS-expressing Vero cells were induced with Dox or were not induced for 24 h. The mRNA (C, upper) and protein levels (C, lower) of NS1 were detected by real-time PCR and western blotting, respectively. The NS1 R38A/K41A and WT viruses from each passage of were obtained from the Tet-On 3G NS1-expressing Vero cells and subjected to plaque assays using MDCK cells to measure the virus titers (D).