Figure 1.
Interferon sensitivity of JUNV in different cell lines.
(A) mouse embryonic fibroblast cells (MEF), (B) Vero cells (ATCC) and (C) human lung carcinoma epithelial A549 cells (ATCC) were treated with IFNs at the indicated concentrations for 16 h. Vero cells and A549 cells were treated with human IFN-α2b (Schering), IFN-β (PBL) or IFN-γ (Sigma), while MEF cells were treated with mouse IFN-β (PBL), respectively. Cells were then infected with VSV, Candid#1 JUNV or Romero JUNV at an MOI of 0.1 PFU/cell. IFNs were supplemented after virus infection. During Romero and Candid#1 JUNV infection, supernatants were collected at 3 days p.i. and assayed for virus production by plaque assay. During VSV infection, supernatants were collected at 16 h.p.i.. Dotted lines indicate the limitation of plaque assay. Data represent the mean of three experiments ±SEM.
Figure 2.
Growth kinetics of JUNV in wild-type MEF and IRF3/7 knockout MEF.
Wild-type MEF cells (A) and IRF3/7 knockout MEF cells (B) were infected by Romero (Rom) and Candid#1 (Can) viruses at an MOI of 0.1. (C) Wild-type MEF cells and IRF3/7 knockout MEF cells were infected by Romero virus at an MOI of 0.001. Supernatants from infected cells were harvested daily and subjected to plaque assay. Note: the titer of Candid#1 virus was below the detection level in both MEFs at an MOI of 0.001. Data represent the mean of triplicates ±SEM. Wt MEF: Wild-type MEF cells. IRF3/7 KO MEF: IRF3/7 knockout MEF cells.
Figure 3.
Immunoblotting analysis of viral protein expression.
MEF cells (A), A549 cells (B) and Vero cells (C) were pretreated with mouse IFN-β (A) or human IFN-β (B and C) at different concentrations as indicated for 16 hr. Cells were then infected with Candid#1 virus at an MOI of 3. Cell lysates were prepared at 1 and 2 days p.i. from MEF cells and A549 cells, or at 2 days p.i. from Vero cells. The viral NP protein was detected with a monoclonal mouse anti-JUNV NP antibody (AG12, BEI) by Western Blotting assay. Equal loading of samples was confirmed by immunoblotting of the same membranes with an antibody to β-actin protein (Santa Cruz). Relative NP protein level in A549 cell samples is shown (B) after densitometry measurement and normalization to the actin protein level.