Fluorescence inversion microscope

The virus titers of CSFV in the cellular supernatant were estimated by IFA. PAMs were inoculated with the supernatant of infected cells in a 96-well plate. Ten groups were prepared with a ten-fold dilution series (10⁻¹–10⁻¹⁰) and incubated for 24 h or 48 h. The negative control was generated by culture with medium without CSFV. All samples were fixed by 1:1 organic fixing liquid (acetone: methanol) at –20 °C for 20 min and permeabilized by 0.1% Triton X-100 at 4 °C for 20 min. After blocking with 5% skimmed milk, permeabilized cells were probed with positive serum against CSFV. Rabbit anti-pig IgG-fluorescein isothiocyanate (FITC) antibody (Sigma) was used following three washes with phosphate-buffered saline (PBS). Immunofluorescence was detected using a fluorescence inversion microscope (Nikon). Negative controls for background staining levels were prepared with mock cells. The viral titers were expressed as TCID₅₀ mL⁻¹.

Four lentiviruses (CMV-IFITMs and shIFITMs), for which titers were sufficiently high for inoculation of PAMs with polybrene (Sigma, St. Louis, MO, USA), were added to the culture medium to enhance the infection rate. The medium was refreshed at 8 h after infection of lentivirus, followed by another 48 h of incubation. Positive cells were selected with puromycin (Thermo Fisher Scientific). Empty CMV and shN served as negative controls. To survey the positive cells that exhibited green fluorescence, a fluorescence inversion microscope (Nikon) was used. The success of overexpression and knockdown of IFITMs was determined by western blot.

IFAs were performed to estimate the virus titers of CSFV in the cellular supernatant. PAMs were inoculated with cellular supernatant in a 96-well culture plate with 10 groups generated by a tenfold dilution series (10⁻¹–10⁻¹⁰) for 48 h, and eight repetitions for each dilution were performed. A negative control was generated by culture with medium without CSFV. These cell samples were fixed by 1:1 fixing liquid (acetone:methanol) at −20 °C for 20 min and permeabilized by 0.1% Triton X-100 in a 4 °C refrigerator for 20 min. Permeabilized cells were incubated with 5% skim milk for 2 h at room temperature, followed by incubation with positive CSFV serum conserved in our laboratory at 4 °C for about 16 h. After washing, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-pig IgG antibody (Sigma) for 2 h. Between each step of the procedure, cells were washed three times using phosphate-buffered saline (PBS) or PBS with Tween 20 in the case of antibody washing. Negative controls were used for background subtraction. FITC-positive cells were observed and counted under a fluorescence inversion microscope (Nikon). The number of positive and negative wells of each dilution was recorded. The viral titers were expressed as 50% tissue culture infectious dose (TCID₅₀)/mL.

CMV plasmid containing ISG15 or pCDH-U6 plasmids carrying shISG15 or shUSP18 were co-transfected into HEK-293T cells with three ancillary plasmids (pGAG, pREV, and pVSV-G) by Turbofect. These cells were cultured in DMEM with 2% FBS for 16 h, and the medium was replaced with DMEM containing 10% FBS, 0.01 mM cholesterol (Sigma), 0.01 mM l-α-phosphatidylcholine (Sigma), 1:1000 diluted Chemically Defined Lipid (Invitrogen, Carlsbad, CA, USA), and 4.0 mM l-glutamine (Invitrogen) followed by another 48 h of incubation. The supernatants were collected and centrifuged at 1500 g to obtain the 5 types of lentiviruses (CMV, CMV-ISG15, shN, shISG15, and shUSP18). Five lentiviruses were added to PAMs in a 6-well culture plate, respectively. The medium was exchanged with fresh medium at 8 h after infection of lentivirus, followed by incubation for another 48 h. CMV and shN served as negative controls. Puromycin (Thermo Fisher Scientific) was used to screen positive cells in complete growth medium at a final concentration of 2 μg/mL. To observe the screened positive cells that exhibited green fluorescence, a fluorescence inversion microscope (Nikon, Tokyo, Japan) was used. Western blot was performed to confirm the overexpression or knockdown of ISGs.

CMV vector containing IFITMs (CMV-IFITM1, CMV-IFITM2, or CMV-IFITM3) or pCDH-U6 vectors carrying IFITM shRNA lentiviruses (shIFITM) were serially co-transfected into HEK-293T cells using three ancillary plasmids, pGAG, pREV, and pVSV-G, by Turbofect. DMEM with 2% FBS was used to culture the transfected cells for 16 h, and then the medium was replaced with DMEM containing 10% FBS, 4.0 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), 0.01 mM cholesterol (Sigma, St. Louis, MO, USA), 0.01 mM L-α-phosphatidylcholine (Sigma), and 1:1000 diluted Chemically Defined Lipid (Invitrogen) followed by another 48 h of incubation. Three types of lentivirus (IFITM1, IFITM2, and IFITM3) in the cell supernatant were collected by centrifugation at 1500× g. Lentivirus titers were estimated in HEK-293T cells. Each of the lentiviruses was inoculated into 10 groups of cells in 96-well plate with a 10-fold dilution series (10⁻¹–10⁻¹⁰), with the CMV vector or shN used as a negative control. The infected cells were detected with a fluorescence inversion microscope (Nikon, Tokyo, Japan), and positive cells manifested by green fluorescence were counted after culture in DMEM with 2% FBS for 48 h. Lentivirus titers were expressed as 50% tissue culture infectious dose (TCID₅₀) mL⁻¹.

CSFV replication (Fig. 4B, Fig. 5C, and Fig. 7B) was measured using an indirect IFA according to a method described previously (17 (link)). Briefly, CSFV-infected cells or inhibitor-treated cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed three times with cold PBS. After fixation, the cells were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and washed three times with PBS. After blocking with 3% BSA for 2 h, the cells were incubated with mouse anti-E2 CSFV antibody (1:200) (Ab-mart) at room temperature for 2 h. After three washes with PBS, the cells were incubated with goat anti-mouse IgG(H+L) (Alexa Fluor 594) antibody (1:200) (catalog number ab150116; Abcam) for 1 h at 37°C. Fluorescence-positive wells were observed using a fluorescence inversion microscope (Nikon, Tokyo, Japan).

An indirect IFA was used for viral titration using a previously described method (17 (link)). Briefly, cells were seeded into 96-well plates and inoculated with CSFV-containing cell supernatants for 72 h in an incubator at 37°C with 5% CO₂. Next, the cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and washed with cold phosphate-buffered saline (PBS) three times. After fixation, the cells were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and washed three times with PBS. After blocking with 3% bovine serum albumin (BSA) for 2 h, the cells were incubated with mouse anti-E2 CSFV antibody (1:200) (Ab-mart) at room temperature for 2 h. After three washes with PBS, the cells were incubated with goat anti-mouse IgG(H+L) (Alexa Fluor 594) antibody (1:200) (catalog number ab150116; Abcam) for 1 h at 37°C. Fluorescence-positive wells were observed and recorded using a fluorescence inversion microscope (Nikon, Tokyo, Japan). Viral titration was determined as the 50% tissue culture infectious dose (TCID₅₀) per milliliter using the method proposed by Reed and Muench (48 ).