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.

In late August 1999, an unusual cluster of cases of meningoencephalitis associated with muscle weakness was reported to the New York City Department of Health. The initial epidemiologic and environmental investigations suggested an arboviral cause.

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.

Introduction of West Nile (WN) virus into the United States in 1999 created major human and animal health concerns. Currently, no human or veterinary vaccine is available to prevent WN viral infection, and mosquito control is the only practical strategy to combat the spread of disease. Starting with a previously designed eukaryotic expression vector, we constructed a recombinant plasmid (pCBWN) that expressed the WN virus prM and E proteins. A single intramuscular injection of pCBWN DNA induced protective immunity, preventing WN virus infection in mice and horses. Recombinant plasmid-transformed COS-1 cells expressed and secreted high levels of WN virus prM and E proteins into the culture medium. The medium was treated with polyethylene glycol to concentrate proteins. The resultant, containing high-titered recombinant WN virus antigen, proved to be an excellent alternative to the more traditional suckling-mouse brain WN virus antigen used in the immunoglobulin M (IgM) antibody-capture and indirect IgG enzyme-linked immunosorbent assays. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WN virus surveillance in the United States and elsewhere.

Through the Advisory Committee on Dengue and other Flavivirus Vaccines, the World Health Organization (WHO) has had a long-standing commitment to facilitate and to guide research and development of vaccines for medically important flaviviruses. Recently, the Paediatric Dengue Vaccine Initiative (PDVI) was formed to accelerate the development, testing, and introduction of dengue (DEN) vaccines worldwide, partnering with WHO in this important public health effort. There are now a variety of DEN vaccines in various stages of the developmental pipeline. In an attempt to make interlaboratory information more directly comparable, WHO with the support of PDVI initiated a program to coordinate the procedures used for the plaque-reduction neutralization test (PRNT). The PRNT is the most common assay used to measure neutralizing antibody. The presence of antibody is believed to be most relevant means of determining protective anti-DEN virus (DENV) immunity. While other neutralizing antibody assays are being considered for use in large-scale vaccine field trials, the PRNT is still considered to be the laboratory standard against which other neutralizing antibody assays should be compared. The need for PRNT coordination has been identified at several consultations between the WHO and PDVI. A more complete version of these guidelines is available on the WHO website: http:sol;sol;www.who.int/immunization/documents/date/en/index.html.