Expression of WNV proteins encoded by plasmid pcDNA-WNV.

<p>(A) Immunofluorescence analysis of E protein expression. HeLa cells were transfected with pcDNA or pcDNA-WNV and, 24 h later, fixed and processed for immunofluorescence using a monoclonal anti-E and a secondary antibody coupled to Alexa Fluor 594. Nuclei were stained with DAPI. Scale bar 20 µm. (B) Analysis of WNV protein expression in the culture medium from 293T or HeLa cells transfected with pcDNA-WNV by enzyme-linked immunodot assay. Both cell lines were transfected with pcDNA or pcDNA-WNV and, 24, 48 or 72 h later, the culture medium was adsorbed onto a nitrocellulose membrane and detected using a monoclonal anti-E or a polyclonal anti-M and the corresponding secondary antibodies coupled to horseradish peroxidase. (C) Western blot analysis of WNV proteins released to the culture medium after transfection with pcDNA-WNV. HeLa or 293T cells were transfected with plasmid pcDNA-WNV and the viral proteins in the culture medium were purified by ultracentrifugation through a 20% sucrose cushion. Proteins were blotted with the antibodies used in panel (B). A control lane containing purified WNV from WNV-infected Vero cells was included. (D) Expression of E protein in the culture medium of HeLa cells stably transfected with pcDNA-WNV (HeLa3-WNV). The E protein content in the culture medium from different passages of HeLa3-WNV cells was determined by an enzyme-linked immunodot assay using a monoclonal antibody against E protein. (E) Analysis by ELISA of the reactivity of WNV antigens released to culture medium of HeLa3-WNV cells. ELISA plates were coated with two-fold serial dilutions of culture medium from HeLa3-WNV cells and incubated either with a monoclonal anti-E or an irrelevant isotype-matched control antibody. Samples were incubated with appropriate secondary antibodies and developed using O-phenylene diamine dihydrochloride. ELISA results were expressed as the absorbance at 492 nm (A492). (F, G) Analysis of the reactivity of viral proteins secreted by HeLa3-WNV with pooled sera from experimentally infected mice (F) or birds (G). ELISA plates were coated with a fixed dilution of HeLa3-WNV culture medium and incubated with two-fold serial dilutions of sera from WNV-infected mice or red-legged partridges and the ELISA was performed as described in (E). Pooled sera from control uninfected animals were included as negative controls.