Serial passaging in vitro generates a Vero cell-adapted coxsackievirus A6 strain with distinct phenotypic characteristics

Figures

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  Figure 1. Biological characteristics of CVA6-3415/XY/CHN/2017 during continuously passaging in vitro. (A) Cytopathic effects (CPEs) were observed at 6 days post-infection (dpi) in Vero cells infected with passages V10, V15, V20, V25, V30, V35, V40, V45, or V50 at a multiplicity of infection (MOI) of 0.01 or mock-infected. (B) Viral titers of V10, V15, V20, V25, V30, V35, V40, V45, or V50 strains were detected by CCID50 assay. (C) The growth curves of recombinants rV10 and rV45 in Vero cells. Vero cells were infected at 0.1 MOI with the indicated recombinants and the cell cultures were harvested at 2, 12, 24, 48, 72, 96 and 120 hpi. Then virus titers were determined by CCID50 assay. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not significant.

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  Figure 2. Enhanced lytic infection and plaque formation of rV45 compared with rV10. (A) CPEs of Vero cells infected with rV10 or rV45 at an MOI of 0.01 were observed at 3 dpi. (B) Plaques formed by rV10 and rV45 in Vero cells. Infected cells were fixed and stained with 1% crystal violet at 96 hpi. (C, D) Vero cells were infected with rV10 or rV45 at an MOI of 0.01. The cells were collected at 1, 2, 3 and 4 dpi, respectively, and subjected to CCK-8 assay (C) for cell viability or LDH release (D) assay for cytotoxicity. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not significant.

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  Figure 3. Enhanced viral entry and release efficiencies of rV45 relative to rV10. (A, B) Viral attachment (A) and internalization (B) assays on Vero cells. Vero cells were infected with equal amounts (100 ng) of FPs of rV10 or rV45, followed by inoculation at 4°C (for attachment) or 37°C (for internalization) for 1 h. Cells were washed, collected and subjected to RT-qPCR for measuring the viral RNA copies. (C) The viral entry ability was calculated as the ratio of internalization to binding in panels A and B. (D, E) Intracellular (D) and extracellular (E) virus progeny were titrated in Vero cells. Vero cells were infected with equal amounts of FPs of rV10 or rV45 (100 ng) for 24 hpi. Cells (for intracellular virus) and supernatants (for extracellular virus) were separately collected and subjected to CCID50 assay for detecting the viral titers. (F) The viral release efficacy was calculated as the ratio of extracellular virus progeny to intracellular virus progeny in panels D and E. Data are presented as mean ± SD. ***p < 0.001; ns, not significant.

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  Figure 4. rV45 exhibiting enhanced interaction with KRM1 and increased KRM1-dependent uncoating compared with rV10. (A, B) HEK-293T cells were transfected with pCAGGS-Flag-KRM1-ect or empty vector for 24 h. Cells were collected and incubated with 5 μg FPs of rV10 or rV45 and anti-Flag magnetic beads at 4°C overnight. Immunoprecipitated complexes were analyzed by western blotting to detect virion–KRM1 interactions (A) or by RT–qPCR to quantify KRM1-associated viral RNA (B). The numbers below the images indicate the IP ratio (co-immunoprecipitated VP1 normalized to immunoprecipitated Flag-KRM1), quantified by densitometric analysis using ImageJ. The IP ratio of rV10 was set to 1.

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  Figure 5. Virulence and immunogenicity of rV10 and rV45 in vivo. (A, B) Three-day-old Kunming mice were intraperitoneally inoculated with 2×104 CCID50 of rV10 or rV45 per mouse. Control animals were inoculated with DMEM medium. Survival rate (A) and mean clinical scores (B) were monitored and recorded daily after infection. (C) Wistar rats were primed and boosted intramuscularly on days 0 and 14 with FPs of rV10 or rV45 at doses of 0.02 μg, 0.1 μg, or 0.5 μg per injection. NtAb titers were determined by using antisera collected on day 28. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

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  Figure 6. Transcriptome analysis of Vero cells infected with rV10 or rV45. (A) Venn diagrams illustrating the number of differentially expressed genes (DEGs) in rV10- and rV45-infected Vero cells. The numbers indicate uniquely and commonly regulated genes among the three comparisons. (B) Stacked bars showing the numbers of up-regulated (red) and down-regulated (green) DEGs in rV10-infected versus mock-infected cells, rV45-infected versus mock-infected cells, and rV45-infected versus rV10-infected cells. (C) KEGG pathway enrichment analysis of genes up-regulated in rV45-infected cells relative to rV10-infected cells. The top 15 enriched pathways are shown. (D) Heatmap showing the expression patterns of representative apoptosis-related genes in rV10- and rV45-infected Vero cells. Gene expression levels were normalized and displayed as row-scaled Z-scores.

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  Figure 7. rV45 inducing apoptosis. (A) Vero cells were infected with rV10 or rV45 (MOI = 1) for 24 h. Cells were stained with Annexin-V and propidium iodide (PI), followed by detecting cell apoptosis via flow cytometry. (B) Vero cells were infected with rV10 or rV45 (MOI = 1). At 24 hpi, the apoptotic response was assessed using a Caspase-3 activity assay. Data represent mean ± SD of fluorescence intensity normalized to the mock group. (C) Vero cells were infected with rV10 or rV45 (MOI = 1) for 24 h. Cells were collected and subjected to western blotting for detecting cleaved caspase 3. β-actin was used as a loading control in western blotting. ***p < 0.001; ns, not significant.

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  Figure 8. Apoptosis is required for efficient replication of rV45. (A-C) Vero cells were pretreated with 20 µM Z-VAD-FMK and then infected with rV10 or rV45 (MOI = 1). At 24 hpi, the cells were collected and subjected to assays of caspase-3 activity (A), western blotting (B) or flow cytometry (C). (D) The apoptotic cell percentage in panel C is displayed in the histogram. (E) Vero cells were pretreated with 20 µM Z-VAD-FMK and then infected with rV10 or rV45 (MOI = 1) for 24 h. Cells were collected and viral titers were determined by CCID50 assay. Data are presented as mean ± SD. ***p < 0.001; ns, not significant.