Fig 2: CXCL8-CXCR1/2 signaling facilitates EV-D68 replication.a, b, d Knockdown of CXCR1/2 suppresses EV-D68 replication in A549 cells. A549-shctl, A549-shCXCL8, A549-shCXCR1, and A549-shCXCR2 cells were infected with EV-D68 (MOI = 0.01), and CPEs (a), viral protein VP1 expression (b), and the viral titer (d) were measured 48 h after infection. c Quantitative analysis of the relative VP1 protein levels in (b). e–g Treatment with the CXCR1/2 inhibitor SX682 inhibits EV-D68 replication. h Quantitative analysis of the relative VP1 protein levels in (g). i SX682 inhibits the activity of the EV-D68 5’UTR. j–l Treatment with the CXCR2 antagonist Danirixin inhibits EV-D68 replication. m Quantitative analysis of the relative VP1 protein levels in (l). n Danirixin inhibits the activity of the EV-D68 5’UTR. Cells treated with DMSO at 12 h, 24 h or 36 h post-infection in (f and k) were set as the control groups. N = 3 (c–f, h–k, m, n) biological replicates. Representative of three biologically independent experiments (a, b, g, l). Data are represented as mean ± SD. Two-tailed t-test (c–e, h–j, m, n) or two-way ANOVA (f and k) was used to assess statistical significance. Scale bar in (a) is 100 μm. Source data are provided as a Source Data file.

Fig 3: VP4 activates CXCL8 via syk-PI3K signaling.a VP0 induces CXCL8 expression. HEK293T cells were transfected with the vector or the expression plasmids for the viral proteins VP1, VP3, VP0, 2A, 2B, 2C, 3A, 3C, and 3D. The relative intracellular CXCL8 mRNA level was measured via qRT‒PCR 24 h after transfection. Cells transfected with vector group was set as control group. b Cells were transfected with increasing amounts of VP0. qRT‒PCR analysis was performed 24 h after transfection to measure the intracellular CXCL8 mRNA level. c VP4 induces CXCL8 expression. Schematic diagram of the cleavage of VP0 into VP4 and VP2. Cells were transfected with the vector or with the VP0, VP2 or VP4 expression plasmid. The relative intracellular CXCL8 mRNA level was measured 24 h post-transfection. d VP4 increases the activity of the CXCL8 promoter. HEK293T cells were co-transfected with CXCL8-promoter-Luc and the vector or the VP0, VP2 or VP4 expression plasmid. Luciferase activity was measured 24 h post-transfection. e, g Mutations in the “YXXI” motif in VP0 (e) and VP4 (g) interfere with the activation of CXCL8. f, h Representative Western blot images depicting VP0 or VP4 expression levels in cells transfected with indicated plasmids, along with corresponding quantitative analysis of the relative VP0 or VP4 protein levels. i Silencing of syk inhibits EV-D68-induced Akt phosphorylation. j Quantitative analysis of the relative pAkt-Ser473 protein levels in (i). k VP4 induces CXCL8 expression through the syk-PIK3CA-Akt pathway. HEK293T-shctl, -shsyk, -shPIK3CA, -shPIK3CB and -shAkt1 cells were transfected with the VP4 expression plasmid. The relative CXCL8 mRNA level was measured 24 h after transfection. l The syk-PI3KA-AKT axis is necessary for EV-D68 replication. A549-shctl, -shsyk, -shPIK3CA, -shPIK3CB, and -shAkt1 cells were infected with EV-D68 (MOI = 0.02) and the viral titers in the culture medium was measured at 24 hpi. N = 3 (a–h, j–l) biological replicates. Representative of three biologically independent experiments (i). Data are represented as mean ± SD. Two-tailed t-test (a–h, j–l) was used to assess statistical significance. Source data are provided as a Source Data file.

Fig 4: EV-D68 infection activates CXCL8 expression in human respiratory cells.a Heatmap visualization of the clustering of differentially expressed genes between EV-D68 (MOI = 0.1)-infected A549 cells and mock-infected cells at 4 h post-infection (hpi). Three replicate samples were set up in each group. b, c Significant increases in CXCL8 transcription and secretion were detected upon EV-D68 infection. A549 cells infected or mock-infected with EV-D68 were collected at the indicated time points. The relative CXCL8 mRNA level was measured via qRT‒PCR (b), and the concentration of secreted CXCL8 was determined via ELISA (c). d Marked upregulation of CXCL8 transcription in human bronchial epithelial cells (HBECs) after EV-D68 infection (MOI = 1). Infected or mock-infected cells at 0 h in (b–d) were set as the control groups. N = 3 (a–d) biological replicates. Data are represented as mean ± SD. Two-tailed t-test (b and d) or two-way ANOVA (c) was used to assess statistical significance. Source data are provided as a Source Data file.

Fig 5: CXCL8 facilitates EV-D68 replication through the MAPK signaling pathway.a EV-D68 infection activates MEK1/2-ERK1/2 signaling in a CXCL8-dependent manner. A549-shctl and A549-shCXCL8 cells were infected with EV-D68 (MOI = 1). The activation of the MEK-ERK signaling pathway was detected within 4 h post-infection. b, c Quantitative analysis of the relative pMEK1/2 or pERK1/2 protein levels in (a). d, f Silencing of either MEK1 (d) or MEK2 (f) inhibits the activating effect of EV-D68 on MAPK signaling. e, g Quantitative analysis of the relative pERK1/2 protein levels in (d and f). The value at 0 min post-infection was set as 1 for each group (b, c, e, g). h, i Silencing of MEK1 (h) or MEK2 (i) inhibits the production of progeny virions. j–l Treatment with the MEK1/2 inhibitor U0126 inhibits EV-D68 replication. m Quantitative analysis of the relative VP1 protein levels in (l). n Cytotoxicity assay with U0126. o U0126 treatment suppresses the function of the enteroviral 5’UTR. p Treatment with U0126 or SX682 inhibits EV-D68 replication in airway organoids. Human airway organoids differentiated for 28 days were infected with EV-D68 (5.0 × 105 TCID50) in the presence or absence of U0126 or SX682. The viral titer in the culture medium was measured 48 h post-infection. N = 3 (b, c, e, g–i, k, m–p) biological replicates. Representative of three biologically independent experiments (a, d, f, j, l). Data are represented as mean ± SD. Two-tailed t-test (b, c, e, g–i, k, m–p) was used to assess statistical significance. Scale bar in (j) is 100 μm. Source data are provided as a Source Data file.