Fig. 6: GW4869 inhibits PRVABC59 strain of ZIKV infection in human astrocytes.

Astrocytes were infected with ZIKV stains PRVABC59 and treated with doses of GW4869 ranging from 2 μM to 10 μM. After 24 h, the cultures were washed and treated with same doses of GW4869 in fresh medium for another 24 h. a Experimental scheme. b–d EVs were isolated from culture supernatants and visualized through NanoSight (b). Quantifications of NanoSight data. ***p < 0.0001 in comparison to ZIKV group (ANOVA, n = 5) (c). The levels of flotillin-2 and Alix in EVs, as well as the levels of GFAP and β-actin in WCL were determined by Western blot (d). e ZIKA RNA was detected in total cellular RNA through real-time RT-PCR. Data were normalized to GAPDH and presented as fold change compared to ZIKV group. f–i Immunocytochemistry of flavivirus antigen was performed on the ZIKV-infected astrocytes. DAPI was used as a nuclear counterstain. Results are representative of three independent experiments. Scale bar: 50 μm. j Quantification of immunofluorescence data was performed with counting flavivirus antigen in infected cells. k ZIKA RNA was detected in total RNA isolated from cell-free supernatants through real-time RT-PCR. l, m After GW4869 treatment, cell-free culture supernatants were added to Vero cells and overlaid with Agar gel. Viral PFU was determined at 4-day post inoculation through crystal violet staining (l). Viral plaques were manually counted and calculated as PFU/ml (m). *p < 0.05; ***p < 0. 001, as compared to the ZIKV group