Induction of antiviral RNA interference by avian coronavirus IBV in PBMCs-Mφ

Figures

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  Fig. 1. Phenotypic differences between QX and M41 in SPF chickens. A Pattern diagram of related phenotype detection in SPF chickens infected with IBV QX and IBV M41. B-C qRT–PCR was used to assess relative IBV-N mRNA expression levels in the trachea, lung, proventriculus, and kidney after infection with IBV-QX (B) and IBV-M41 (C) at the 24 and 48 h time points. Data show expression calculated relative to that of β-actin and are presented as the fold changes relative to that of the blank group (an equal amount of PBS was added as a control), normalized to 1. D IBV QX and M41 loads in the trachea, lung, proventriculus, and kidney detected by IHC at the 48 h infection time point (original magnification: 40×). The data are shown as the mean ± SD; n = 3. The differences were not significant at P ≥ 0.05 (ns) or significant at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***)

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  Fig. 2. QX and M41 induce different RNAi phenomena in PBMCs-Mφ. A-B Viral loads in PBMCs, red cells, and other cells were determined using qRT–PCR after IBV-QX (A) and IBV-M41 (B) infection for 24 and 48 h. C Microscopic pictures of PBMCs-Mφ on the indicated culture days (Days 1–3), and the black arrows indicate PBMCs-Mφ. Original magnification scale bar, 50 μm. D FCM results of macrophage sorting based on the PE-KUL01 antibody from the peripheral blood of chickens. E MHC II expression levels in PBMCs-Mφ were detected by flow cytometry after infection with IBV-QX and IBV-M41 at 24 and 48 h. The mean fluorescence intensity (MFI percentage) of MHC II was quantified, as shown on the grey side. F Confocal microscopy was used to examine PBMCs-Mφ infected with IBV-QX and IBV-M41 at 24 and 48 h. The cells were labelled with DAPI (blue) and an IBV N protein antibody (red). Scale bar, 10 μm. G Fluorescence intensity analysis of the data in Fig. 2F. H Viral titres in IBV QX- and M41-infected PBMCs-Mφ from 12–60 h were detected using the TCID50 method; n = 3 per group. The data are shown as the mean ± SD; n = 3. The differences were not significant at P ≥ 0.05 (ns) or significant at P < 0.05 (*) or…

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  Fig. 3. Changes in IFN and RNAi pathways after IBV infection. A-B PBMCs-Mφ were infected with IBV-QX (A) and IBV-M41 (B) for 24 and 48 h, with uninfected cells used as the blank group. qRT–PCR was used to assess the relative mRNA expression levels of MDA5, MAVS, TLR3, IFN-α, IFN-β, and IFN-γ. C-D PBMCs-Mφ were infected with IBV-QX (C) and M41 (D) for 24 and 48 h, and qRT–PCR was used to assess the relative mRNA expression levels of Dicer and Ago2. E--F western blotting was used to analyse Dicer and Ago2 expression in PBMCs-Mφ infected with IBV-QX (E) or IBV-M41 (F) at 24 and 48 h. The right panel shows the results of the grey value analysis. The data are shown as the mean ± SD; n = 3. The differences were considered significant at P < 0.05 (*), P < 0.01 (**), or not significant at P ≥ 0.05 (ns)

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  Fig. 4. Small RNA sequencing and vsiRNA analysis. A PBMCs-Mφ were infected with IBV-QX and IBV-M41 for 24 h, and the uninfected cells were used as the blank group. The RNA samples were subjected to small RNA sequencing, and the differences in lncRNAs, circRNAs, and miRNAs between the QX- and M41-infected PBMCs-Mφ groups were analysed. B-C Mechanistic diagram of vsiRNA screening (B) and the number of (18–25 nt) vsiRNAs (C). D-E Position display diagram of 18–25 nt vsiRNA reads (D) and 21–23 nt vsiRNA sequences (E) in the IBV M41 and QX genomes

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  Fig. 5. A Relative mRNA expressions of IBV-N after IBV (QX and M41) infected BMDCs. B Statistical analysis of vsiRNAs after 24 h of IBV infection in BMDCs. C Visualization of the 21-23 nt vsiRNAs distribution and the enrichment region in the genome.

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  Fig. 6. PS-vsiRNA construction and antiviral validation. A-B Map of psilencer4.1-vsiRNA (PS-vsiRNA) plasmid construction (A) and identification of BamHI/HindIII enzyme digestion (B), including PS-Q10, PS-Q16, PS-M10, and PS-M16. C-E PBMCs-Mφ were transfected with PS-vsiRNA plasmids (PS-Q10, PS-Q16, PS-M10, and PS-M16) for 24 h and subjected to infection with IBV-QX and IBV-M41 (C). qRT–PCR was used to detect Dicer, Ago2 (D), and IBV-N (E) mRNA expression. Notably, the M41 group (PS-M10 and PS-M16) and the QX group (PS-Q10 and PS-Q16) were infected with IBV (QX and M41). F western blotting was used to analyse the expression levels of β-actin, IBV-N, and Dicer in the IBV-infected PBMCs-Mφ group. The right panel shows the grayscale analysis results. The data are shown as the mean ± SD; n = 3. The differences were not significant at P ≥ 0.05 (ns) or significant at P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), or P < 0.0001 (****)

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  Fig. 7. The QX-NSP10/NSP16 complex is a potential VSR protein. A FCM was used to analyse the changes in MHC II expression after transfection with PC-Q10, PC-Q16, PC-M10, or PC-M16. MFI shows the intensity of MHC II, where CpG and LPS are positive controls and PC is a negative control. B-D The relative mRNA expression levels of IBV-N (B), IFN-γ (C), Dicer, and Ago2 (D) were assessed by qRT–PCR after PBMCs-Mφ were transfected with PC, PC-M10 and PC-M16 (the M41 group), or PC-Q10 and PC-Q16 (the QX group) and infected with IBV (QX and M41). The data are shown as the means ± SDs; N = 3. The difference value analysis results are as follows: ****P < 0.0001, ***P < 0.001, ** P < 0.01, *P < 0.05, ns P > 0.05