SHFL Post-Transcriptionally Restricts Coxsackievirus A16 In Vitro and In Vivo

Figures

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  Figure 1. Induction of Shiftless (SHFL) by CVA16 infection and generation of SHFL knockout RD cells. (A) Relative mRNA expression levels of selected interferon-stimulated genes (ISGs) in RD cells following CVA16 infection, as determined by quantitative RT-PCR. (B) SHFL mRNA expression in RD cells infected with CVA16 at the indicated multiplicities of infection (MOIs), measured by quantitative RT-PCR. (C) SHFL mRNA expression in RD cells treated with exogenous IFN-β at the indicated concentrations. (D) Schematic illustration of the CRISPR–Cas9 strategy used to generate SHFL knockout (KO) RD cells. (E) Cell viability or growth characteristics of parental RD cells and monoclonal SHFL KO cell lines following single-cell sorting. (F) Western blot analysis of SHFL protein expression in parental RD cells and the selected SHFL KO clone (#19). Data are presented as mean ± standard deviation (SD; n = 3). ** p < 0.01, and *** p < 0.001.

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  Figure 2. Analysis of CVA16 infection in SHFL knockout and wild-type RD cells. (A) Cytopathic effects observed in SHFL knockout (SHFL KO), wild-type (WT), and mock-infected RD cells at 24 h post-infection (hpi). Scale bars represent 200 μm. (B) Plaque assay analysis of CVA16 produced from SHFL KO and WT RD cells. Culture supernatants collected from infected cells were serially diluted and subjected to plaque formation assays. (C) One-step growth curve of CVA16 in SHFL KO and WT RD cells determined by TCID50 assay at the indicated time points post-infection. The shaded background highlights the time window with the greatest differences between groups. (D) One-step growth curve based on viral RNA quantification. Relative CVA16 viral RNA levels were measured by quantitative RT-PCR at the indicated time points post-infection. (E) Immunofluorescence staining of CVA16-infected SHFL KO and WT RD cells at 24 h post-infection. Scale bars represent 200 μm. (F) Rescue experiment in SHFL KO RD cells. Cells were transiently transfected with a SHFL expression plasmid or empty vector and subsequently infected with CVA16. Viral replication was assessed by quantitative RT-PCR analysis of viral RNA levels.…

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  Figure 3. Disease progression in SHFL knockout and wild-type neonatal mice following CVA16 infection. (A) Body weight changes in six-day-old SHFL knockout (SHFL KO) and wild-type (WT) neonatal mice following CVA16 infection, expressed as a percentage of initial body weight. The shaded background indicates the time window with the most evident differences, and the vertical dashed line marks the divergence point. (B) Survival curves of six-day-old SHFL KO and WT neonatal mice following CVA16 infection. (C) Clinical scores of six-day-old SHFL KO and WT neonatal mice monitored daily after CVA16 infection. (D) Body weight changes in three-day-old WT neonatal mice following CVA16 infection. The shaded background indicates the time window with the most evident differences, and the vertical dashed line marks the divergence point. (E) Survival curves of three-day-old WT neonatal mice following CVA16 infection. (F) Clinical scores of three-day-old WT neonatal mice monitored daily after CVA16 infection. (G) Representative image of a mock-infected neonatal mouse showing normal posture and limb movement. (H) Representative image of a three-day-old neonatal mouse following CVA16 infection, showing mild…

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  Figure 4. Tissue viral loads in SHFL knockout neonatal mice following CVA16 infection. Viral titers in indicated tissues of SHFL knockout (SHFL KO) neonatal mice at 1, 3, and 5 days post-infection (dpi). Tissues analyzed included skeletal muscle, lung, brain, spinal cord, liver, heart, spleen, and kidney. Viral loads were quantified by TCID50 assay using RD cells, as described in the Materials and Methods. Data are presented as mean ± standard deviation (SD; n = 5). * p < 0.05, ** p < 0.01, and *** p < 0.001.

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  Figure 5. Histological analysis of tissues from mock-infected and CVA16-infected SHFL knockout mice. (A,B) Hematoxylin and eosin (H&E) staining of brain sections from mock-infected (A) and CVA16-infected (B) SHFL knockout (SHFL KO) mice. Low-magnification images are shown on the left, with corresponding high-magnification views of the boxed areas on the right. (C,D) H&E staining of spinal cord sections from mock-infected (C) and CVA16-infected (D) SHFL KO mice. (E,F) H&E staining of heart sections from mock-infected (E) and CVA16-infected (F) SHFL KO mice. (G,H) H&E staining of liver sections from mock-infected (G) and CVA16-infected (H) SHFL KO mice. (I,J) H&E staining of spleen sections from mock-infected (I) and CVA16-infected (J) SHFL KO mice. (K,L) H&E staining of lung sections from mock-infected (K) and CVA16-infected (L) SHFL KO mice. (M,N) H&E staining of kidney sections from mock-infected (M) and CVA16-infected (N) SHFL KO mice. (O,P) H&E staining of skeletal muscle sections from mock-infected (O) and CVA16-infected (P) SHFL KO mice. Arrows indicate regions with severe pathological damage. Scale bars represent 200 μm in low-magnification images and 50 μm in high-magnification…

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  Figure 6. Immunohistochemical analysis of CVA16 antigen in tissues from SHFL knockout mice. (A,B) Immunohistochemical staining of lung sections from mock-infected (A) and CVA16-infected (B) SHFL knockout (SHFL KO) mice. (C,D) Immunohistochemical staining of skeletal muscle sections from mock-infected (C) and CVA16-infected (D) SHFL KO mice. (E,F) Immunohistochemical staining of spinal cord sections from mock-infected (E) and CVA16-infected (F) SHFL KO mice. (G,H) Immunohistochemical staining of brain sections from mock-infected (G) and CVA16-infected (H) SHFL KO mice. Representative low-magnification images are shown on the left, with corresponding high-magnification views of the boxed areas on the right. Arrows indicate CVA16 antigen–positive immunostaining. Scale bars represent 200 μm in low-magnification images and 50 μm in high-magnification images. Immunostaining was performed using a polyclonal anti-CVA16 antiserum; signal intensity should therefore be interpreted cautiously, particularly in tissues with low viral burden.

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  Figure 7. Nissl staining analysis of spinal cord and brain tissues from mock-infected and CVA16-infected SHFL knockout mice. (A,B) Nissl staining of spinal cord sections from mock-infected (A) and CVA16-infected (B) SHFL knockout (SHFL KO) mice. (C) Quantification of Nissl-positive neurons in the spinal cord. (D,E) Nissl staining of brain sections from mock-infected (D) and CVA16-infected (E) SHFL KO mice. (F) Quantification of Nissl-positive neurons in the brain. Data are presented as mean ± standard deviation (SD). ** p < 0.01, and *** p < 0.001. Scale bars represent 100 μm in low-magnification images and 20 μm in high-magnification images.

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  Figure 8. Transcriptomic analysis of WT and SHFL knockout cells following CVA16 infection. (A) Volcano plots showing differentially expressed genes in WT cells following CVA16 infection. (B) Volcano plots showing differentially expressed genes in SHFL knockout (SHFL KO) cells following CVA16 infection. (C) Venn diagram showing the overlap of upregulated and downregulated differentially expressed genes between WT and SHFL KO cells after CVA16 infection. (D) Heatmap showing hierarchical clustering of representative differentially expressed genes across WT and SHFL KO samples. (E) Gene Ontology (GO) enrichment analysis of selected differentially expressed genes. (F) Summary of enriched Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways based on differentially expressed genes. n = 3 biological replicates per group.