Figure 5.

ORF3 inhibits Poly I:C mediated type I interferon production and induction. HEK293T cells were transfected with Poly I:C and ORF3. Cells transfected with empty plasmid were used as mock transfection. At 24 hpt, undiluted and serial 2-fold dilutions (1/2 and 1/4) of supernatants were collected and incubated in PK-15 cells for 24 hrs. After incubation, cells were infected with VSV-mCherry. Spreading of the virus in the cell monolayer was monitored by the detection of mCherry fluorescence with fluorescence microscopy (A). (B) Undiluted and serial 2-fold dilutions (1/2, 1/4 and 1/8) of supernatants (n = 3) were added into 96-well plate containing HEK-Blue^TM IFN-α/β cell suspension. After incubation for 24 hrs, induced HEK-Blue^TM IFN-α/β cell supernatants were used to measure type I IFN induction through JAK-STAT pathway activation by a secreted embryonic alkaline phosphatase (SEAP) detection following the manufacturer’s protocol. The SEAP levels were quantified using a spectrophotometer at 655 nm. Error bars indicated mean ± standard error of means of absorbance values. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.