1. Preparation of A549 cells and JUNV infection

1. Seed 2 x 10⁵ human lung epithelial A549 cells onto poly-D-lysine (PDL) coated glass coverslips in 12-well plates at 24 hours prior to infection.
2. Prepare aliquots of 150 μL of JUNV¹³ at a multiplication of infection (MOI) of 1.0 plaque forming unit per cell diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) media supplemented with 2% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S).
3. Remove media from cell culture. Add virus inoculum on each well containing coverslip and incubate for 1.5 h at 37 °C. Shake the plates every 15 min.
4. Remove the virus inoculum, add 1 mL of DMEM supplemented with 5% FBS and 1% P/S. Incubate the plate at 37 °C for desired time point.

2. Fixation and immunostaining

1. Aspirate the media. Rinse the cells by adding 1 mL of phosphate buffered saline (PBS) supplemented with calcium and magnesium to each well.
2. Remove PBS. Add 1 mL of methanol (MeOH) pre-chilled at -20 °C and incubate at -20 °C or on dry ice for 15 min.
3. Remove MeOH.
4. Add 1 mL of PBS to each well, and wash samples at 4 °C with gentle rocking for 5 min. Repeat the wash for total of 4 times.
5. Wash the fixed cells on coverslips in 1 mL of 0.2% t-octylphenoxypolyethoxyethanol for 5 min at 4 °C, with gentle rocking.
6. Add 1 mL of PBS to each well. Wash samples at 4 °C for 5 min with gentle rocking. Repeat the washing step for total of 4 times.
7. To detect dsRNA and MDA5, incubate samples in 200 μL of primary antibodies diluted in 3% bovine serum albumin (BSA). Dilute the anti-dsRNA 9D5 antibody at a 1:2 dilution and dilute the anti-MDA-5 antibody at 1:250. Incubate with gentle rocking at 4 °C overnight.
8. Remove primary antibodies and wash each well with 1 mL of PBS for 5 min with gentle rocking at RT. Repeat this wash four more times.
9. Add 200 μL of secondary antibodies (1:2,000 dilution) diluted in 3% BSA and incubate at RT for 1 h.
10. Remove secondary antibodies and wash each well with 1 mL of PBS for 5 min with gentle rocking at RT. Repeat this wash four more times.
11. To detect JUNV nucleoprotein (NP) and RIG-I, add 200 μL of conjugated antibodies diluted in 3% BSA. Dilute the conjugated anti-JUNV NP (AG-12) at 1:1,000 and incubate samples for 2 h at RT with gentle rocking. Dilute the conjugated anti-RIG-I antibody at 1:500 and incubate at 4 °C overnight with gentle rocking.
12. Remove conjugated antibodies and wash each well with 1 mL of PBS for 5 min with gentle rocking at RT. Repeat this wash four more times.
13. Counterstain the coverslips with DAPI (1:1,000) for 3 min with gentle rocking at RT.
14. Wash the coverslips 3 times, each time for 5 min in 1 mL of 0.5% t-octylphenoxypolyethoxyethanol with gentle rocking at RT.
15. Wash twice in 1 mL of PBS for 5 min each time with gentle rocking at RT.
16. Wash once in 1 mL of ddH₂O for 1 min at RT, rocking gently.
17. Mount coverslips onto glass slides using mounting media. Let cure overnight.
18. Seal the slides with nail polish and air dry for 1 h.
19. Image on confocal microscope with the 60x/1.42 numerical aperture oil immersion lens using the same laser emissions for each sample.
20. When analyzing the data, if necessary, make adjustments for brightness and contrast using the same linear adjustment for all samples.