In late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows (Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo (Phoenicopterus chilensis), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein-specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

The specific mechanisms by which antibodies neutralize flavivirus infectivity are not completely understood. To study these mechanisms in more detail, we analyzed the ability of a well-defined set of anti-dengue (DEN) virus E-glycoprotein-specific monoclonal antibodies (MAbs) to block virus adsorption to Vero cells. In contrast to previous studies, the binding sites of these MAbs were localized to one of three structural domains (I, II, and III) in the E glycoprotein. The results indicate that most MAbs that neutralize virus infectivity do so, at least in part, by the blocking of virus adsorption. However, MAbs specific for domain III were the strongest blockers of virus adsorption. These results extend our understanding of the structure-function relationships in the E glycoprotein of DEN virus and provide the first direct evidence that domain III encodes the primary flavivirus receptor-binding motif.The flavivirus E glycoprotein is the primary antigen inducing protective immunity, is essential for membrane fusion, and mediates binding to cellular receptors. Therefore, this protein directly affects host range, cellular tropism, and, in part, the virulence of these viruses (17, 18). The crystal structure of the ectodomain of the tick-borne encephalitis (TBE) virus E-glycoprotein homodimer was recently solved at high resolution (16). Multiple lines of evidence indicate that this E-glycoprotein structure is strongly conserved across the Flaviviridae (16). This protein contains three structural domains. The central domain, domain I (DI), contains predominately type-specific nonneutralizing epitopes and is theorized to be the molecular hinge region involved in low-pH-triggered conformational changes (19). The dimerization domain, domain II (DII), makes important contacts with itself in the homodimer, is involved in virus-mediated membrane fusion, and contains many cross-reactive epitopes eliciting neutralizing and nonneutralizing monoclonal antibodies (MAbs) (16,19). Domain III (DIII) is characterized by an immunoglobulin-like structure containing the most distal projecting loops from the virion surface. It contains multiple type-and subtype-specific epitopes eliciting only virus-neutralizing MAbs and has been hypothesized to contain the host cell-binding antireceptor (16,18,19). As part of our ongoing research to elucidate the structure-function relationships of the dengue (DEN) virus E glycoprotein, we have assessed the ability of a well-characterized panel of E-glycoprotein-specific MAbs to block virus adsorption to Vero cells. These results provide the first direct evidence that E glycoprotein DIII encodes a receptor-binding motif.

The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.