Fig. 5
Effect of rMuIFNλ3 pre-treatment on B16F10 cells against A/PR/8/1934 (H1N1) infection. Representative images showing comparative CPEs exhibited by virus-infected B16F10 cells (1.0 MOI) pre-treated with different forms of MuIFNλ3. The images were captured in the Nikon TS100 inverted light microscope (Nikon) at ×20 magnification (a–e). Indirect immunofluorescence assay (IFA) showing a significant reduction of viral NP (in green) in the cells pre-treated with MuIFNλ3 secreted by rL. lactis or expressed by E. coli (i, j) compared to untreated/WT L. lactis treated infected cells (g, h). For nuclear staining 4′,6-diamidino-2-phenylindole-dihydrochloride (DAPI) (in blue) was used. All images were visualized in the Leica SP8 confocal microscope using oil immersion ×63 objective (NA 1.4), DAPI, and FITC filter. Blue fluorescence corresponds to DAPI staining of the nucleus, and green fluorescence corresponds to the H1N1 viral NP protein. Scale Bar: 50 µm (f, j). Percentage of cell survival by standard MTT assay showing significantly higher cell survival when cells were pre-treated with different forms of MuIFNλ3 (rL. lactis or rMuIFNλ3) against H1N1 virus infection. Each bar represents the mean of percent cell survival ± SE of three independent experiments performed in triplicates. Asterisks (**) in (p < 0.01) indicate statistically significant differences (control vs. treatment) (k). Absolute quantification of viral M-gene transcripts by RT-qPCR showing substantial reduction in M-gene copies in infected cells pre-treated with MuIFNλ3 (rL. lactis and rMuIFNλ3) compared to the control groups (Virus only and WT L. lactis). Each bar represents the mean of the M-gene transcript number ± SE of two independent experiments performed in duplicates. Asterisks (*) indicate statistically significant differences (p ≤ 0.05) with respect to the control (l). Viral haemagglutination (HA) assay to detect the virus titer in the culture supernatant of infected B16F10 indicates a significant reduction in HA titer in MuIFNλ3 pre-treated cells compared to controls. Two-fold serial dilutions of each sample were incubated with 0.5% chicken RBC (cRBC). The reciprocal of the highest dilution of the sample that causes complete hemagglutination of cRBC was considered HA titer (m). Each bar represents the HA titer of different treatment setups and is compared to the control, showing a significant reduction in HA titer in the case of MuIFNλ3 treated cells (n)