Several studies have reported the usefulness of phage-display applications for mapping epitopes of flaviviruses [[22–25]]. The aim of our study was to identify WNV-specific and/or JEV serocomplex-specific B-cell epitopes in NS1 using phage display technology. The information provided by this study will facilitate the development of diagnostic tools for the specific serological diagnosis of WNV infection, and will contribute to learn more the rational design of vaccines by furthering understanding

of the antigenic structure of NS1. Results Production of recombinant NS1 Recombinant WNV NS1 was successfully expressed in E. coli TB1 cells, predominantly as soluble protein, after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was recognized by WNV-positive equine serum in Western blot (WB) (Figure 1, lane 1). Figure 1 WNV-positive equine sera high throughput screening assay recognize recombinant NS1. Binding of antibodies from WNV-positive equine serum

to recombinant NS1 (lane 1) and MBP-tag (lane 2) by Western blot. M, PageRuler™ Prestained Protein Ladder (Fermentas, Canada). Production and characterization of NS1-specific mAbs Purified protein was used to immunize BALB/c mice. After cell fusion and screening, several hybridoma cell lines were obtained which produced NS1-specific mAbs. Among them two cell lines were selected for their strongest reactivity against recombinant NS1 using indirect ELISA (data not shown), WB (Figure 2a), and against native NS1 in IFA using WNV antigen slides (Figure 2b). Further characterization of the specificity of the two mAbs by IFA, demonstrated that the mAb 3C7 reacted with WNV, but did not react with JEV, DENV1-4, Tipifarnib in vivo Yellow fever virus (YFV) and Tick-borne encephalitis virus (TBEV), whereas mAb 4D1 reacted with both WNV and JEV, but did not react

with other non-JEV serocomplex flaviviruses (Figure 2b). Figure 2 Reactivity of mAbs with recombinant NS1 and C6/36 cells infected with flaviviruses. (a) Western blot analysis of mAbs 3C7 (lanes 1, 2) and 4D1 (lanes 3, 4) against recombination NS1 (lane 1, 3) and MBP-tag (lane 2, 4). M, PageRuler™ Prestained Protein Ladder (Fermentas, Canada). (b) Pattern of immunofluorescence Dimethyl sulfoxide produced by anti-NS1 mAbs on antigen slides which were prepared on porous slides using C6/36 cells infected with different flaviviruses. Panels 1-8: reactivity of mAb 3C7 with cells infected with WNV (panel 1), JEV (panel 2), DENV1 (panel 3), DENV2 (panel 4), DENV3 (panel 5), DENV4 (panel 6), YFV (panel 7), and TBEV (panel 8). Panels 11-18: reactivity of mAb 4D1 with cells infected with WNV (panel 11), JEV (panel 12), DENV1 (panel 13), DENV2 (panel 14), DENV3 (panel 15), DENV4 (panel 16), YFV (panel 17), and TBEV (panel 18).