Fig. 5: Sphingosine suppressed ZIKV infection by affecting membrane fusion.

a Schematic diagram of the study design. b,c Effects of sph addition in different ways in ZIKV-infected cells. ZIKV mRNA levels were examined using qPCR (b; n = 3 biologically independent cell samples; two-sided multiple unpaired t-test), and the protein levels were quantified using western blot (c) at 48 h post infection. Con.: DMSO treatment. d The attachment assay of ZIKV-infected C6/36 cells. Prechilled C6/36 cells were incubated with ZIKV plus 0 or 10 μM sph for 30 min, and RNA was then extracted from cells (n = 6 biologically independent cell samples; two-sided unpaired t-test). Con.: DMSO treatment. e The insertion assay. ZIKV was mixed with liposomes at pH 5.75 with or without 50 µM sph for 30 min. The mixture was then separated into top, middle, and bottom fractions by floating on a sucrose gradient. Samples at pH 8.0 were used as the negative control (n = 3 biologically independent mixtures; two-way ANOVA test followed by Tukey’s post hoc tests). f The fusion assays of ZIKV-infected Vero cells. Infected cells were detected by immunofluorescence (n = 3 biologically independent cell samples). The IC₅₀ of sph against ZIKV was calculated. Infections observed at pH 6.0 and pH 7.9 were used as positive and negative controls, respectively. g Identifying sph levels in the midguts of Aedes mosquitoes using HPLC-Q-TOF (n = 3 biologically independent midguts; one-way ANOVA test followed by Tukey’s post hoc tests). UGAL: lab-adapted Ae. aegypti (UGAL/Rockefeller strain). AAL (left panel): lab-adapted Ae. albopictus (Jiangsu strain). Antibiotic-treated AAL (left panel): lab-adapted Ae. albopictus (Jiangsu strain) with antibiotic treatment. AAEL: field-derived Ae. aegypti (Haikou strain). AAL (right panel): field-derived Ae. albopictus (Haikou strain). Antibiotic-treated AAL (right panel): field-derived Ae. albopictus (Haikou strain) with antibiotic treatment. Data are represented as mean ± SEM. The experiment was repeated twice with similar results. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant. The original scans for Fig. 5c were provided in Supplementary Fig.7. Source data are provided as a Source Data file.