title: West Nile virus: characterization and diagnostic applications of monoclonal antibodies.
abstract: BACKGROUND
  Diagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs.
  
  
  METHODS
  Six MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity.
  
  
  RESULTS
  All MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys → Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser → Ile) or E278 (Thr → Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs.
  
  
  CONCLUSIONS
  MAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization.
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 9, 25124 Brescia, Italy. davide.lelli@izsler.it
authors.forename: Davide
authors.initials: D
authors.lastname: Lelli
authors.forename: Ana
authors.initials: A
authors.lastname: Moreno
authors.forename: Emiliana
authors.initials: E
authors.lastname: Brocchi
authors.forename: Enrica
authors.initials: E
authors.lastname: Sozzi
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Capucci
authors.forename: Elena
authors.initials: E
authors.lastname: Canelli
authors.forename: Ilaria
authors.initials: I
authors.lastname: Barbieri
authors.forename: Herve
authors.initials: H
authors.lastname: Zeller
authors.forename: Paolo
authors.initials: P
authors.lastname: Cordioli
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D002645:Chickens; D019411:Clinical Laboratory Techniques; D003429:Cross Reactions; D018604:Epitope Mapping; D018397:Equidae; D006801:Humans; D007118:Immunoassay / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods; D014901:West Nile Fever / Q000175:diagnosis* / Q000821:virology; D014902:West Nile virus / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral