Transcriptome analysis of African swine fever virus I9R-mediated modulation of host antiviral immunity

Figures

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  Fig. 1. ASFV I9R is an early transcription gene. (A) Amino acid sequence alignment of ASFV I9R. The amino acid sequences of I9R from 10 African swine fever virus strains were compared to characterize the pattern of multi-residue substitutions by MEGA5.1 software. (B) The expression levels of I9R, CP204L, and B646L mRNA. RT-qPCR was performed on I9R, CP204L, B646L and GAPDH using specific primers to detect the average cycle thresholds of ASFV-WT infected primary porcine alveolar macrophages (MOI of 1.0) at 0, 3, 6, 9,12,24 and 48 hpi. (C) Localization of pI9R protein in HEK293T cells. Under confocal microscopy, HEK293T cells were transfected with pCGAAS-I9R-Flag. At 24 hpi, the cells were incubated with mouse anti-flag and Alexa Fluor 488(green) conjugated secondary antibodies, respectively. The nuclei (blue) were stained with 4′, 6-diamino-2-phenylindole (DAPI). bar, 10 μm.

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  Fig. 2. Construction of ASFV-∆I9R. (A) Schematic diagram of the construction of I9R deletion mutant. (B) Infection of PAMs with purified ASFV-∆I9R, scale, 150 µm. (C) PCR amplification confirmed the deletion of the ASFV I9R gene. (D) Sequencing results for the region covering the homologous recombination site of the ASFV-ΔI9R strain. (E) Cells were infected with ASFV-WT or ASFV-∆I9R (MOI = 1) respectively, and samples were collected at 6, 12, 24 and 48 hpi after infection respectively. RNA was extracted by quantitative reverse transcription PCR (qRT-PCR) and the transcriptional level of the I9R gene was detected using I9R primers.

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  Fig. 3. Deletion of I9R does not affect ASFV replication in PAMs. (A) HAD50 assay. PAMs were infected with ASFV-ΔI9R or the parental ASFV-WT strain. At 24 hpi, whole blood from healthy pigs was added. Hemadsorption rosettes formed by red blood cells were observed in PAMs infected with ASFV-WT or ASFV-ΔI9R. In addition, green fluorescence was detected in ASFV-ΔI9R-infected cells, indicating the expression of EGFP. No hemadsorption or fluorescence signal was observed in uninfected control cells. (B) Growth kinetics analysis. PAMs were infected with ASFV-WT or ASFV-ΔI9R at MOI of 1. Samples were collected at 3, 6, 9, 12, 24, 36, 48, and 72 hpi. Viral titers were determined using the HAD₅₀ assay. (C) Viral genome replication analysis. PAMs were infected with ASFV-WT or ASFV-ΔI9R (MOI = 1), and samples were collected at 3, 6, 9, 12, 24, 36, 48, and 72 hpi. Viral genome replication levels were determined by quantitative PCR.

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  Fig. 4. GO and KEGG analysis of ASFV-∆I9R-infected PAMs through RNA-seq. (A) GO analysis of ASFV-∆I9R vs. ASFV-WT at 18 hpi. (B) GO analysis of ASFV-∆I9R vs. ASFV-WT at 36 hpi. (C) KEGG analysis of ASFV-∆I9R vs. ASFV-WT at 18 hpi. (D) KEGG analysis of ASFV-∆I9R vs. ASFV-WT at 36 hpi.

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  Fig. 5. Analysis of differentially expressed genes. (A) Volcano plot showing DEGs between ASFV-ΔI9R infected and ASFV-WT infected PAMs at 18 hpi. (B) Volcano plot showing DEGs between ASFV-ΔI9R infected and ASFV-WT infected PAMs at 36 hpi. (C) Venn diagram showing DEGs between ASFV-ΔI9R infected and ASFV-WT infected PAMs at 18 and 36 hpi. The pink area represents DEGs identified in ASFV-ΔI9R infected vs. ASFV-WT infected PAMs at both 18 and 36 hpi; the red area represents DEGs identified only at 18 hpi; and the blue area represents DEGs identified only at 36 hpi.

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  Fig. 6. Validation of RNA-seq results by RT-qPCR. (A and B) PAMs were either uninfected or infected with ASFV-WT or ASFV-ΔI9R at MOI of 1. Cells were collected at 18 and 36 hpi, and total RNA was extracted for RT-qPCR analysis to determine gene expression levels. Data are presented as mean ± SD. Statistical significance was analyzed using one-way analysis of variance (ANOVA) Student’s t-test. **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (p > 0.05).

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  Fig. 7. Deletion of I9R suppresses host innate immune responses during ASFV infection. (A and B) PAMs were infected with ASFV-WT or ASFV-ΔI9R at 1 MOI. At 18 and 36 hpi, cells were treated with 400 ng/mL IFN-β for 6 hpi. Total RNA was extracted using TRIzol reagent, and the transcription level of ISG12A was determined by RT-qPCR. Error bars represent the standard error of the mean. (C) PAMs were infected with ASFV-WT or ASFV-ΔI9R (MOI = 1). At 18 and 36 hpi, cells were treated with 400 ng/mL IFN-β for 6 hpi, followed by Western blot analysis using the indicated antibodies. Statistical significance was analyzed using one-way analysis of variance (ANOVA). **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (p > 0.05).