Use of a Recombinant Envelope Protein Subunit Antigen for Specific Serological Diagnosis of West Nile Virus Infection

Abstract

Serological diagnosis of West Nile virus (WNV) infection is complicated by extensive antigenic crossreactivity with other closely related flaviviruses, such as St. Louis encephalitis virus.Here we describe a recombinant, bacterially expressed antigen equivalent to structural domain III of the WNV envelope protein that has allowed clear discrimination of antibody responses to WNV from those against other related flaviviruses in indirect enzyme-linked immunosorbent assays using standardized control antisera and field-collected samples.Since 1999, the United States has experienced annual epidemics of disease in humans and animals caused by West Nile virus (WNV) over an expanding geographical range. To date during 2003, WNV has been isolated in 46 states and the District of Columbia, and more than 8,500 cases of human disease, resulting in 199 deaths, have been reported (1). Outbreaks of WNV disease with neurological manifestations have also been reported in Eastern Europe, North Africa, and Israel since the mid-1990s (reviewed in reference 17). WNV is clearly an emerging and significant public health problem. Research priorities to limit the impact of WNV include the development of more-specific rapid diagnostic assays (5, 18).WNV is a member of the Japanese encephalitis (JE) virus group of the genus Flavivirus, family Flaviviridae; other members include Japanese encephalitis virus (JEV), found throughout Asia, St. Louis encephalitis virus (SLEV), found in the Americas, and Murray Valley encephalitis virus (MVEV), found in Australia and New Guinea. These viruses have a similar ecology and are antigenically related to WNV, and their cocirculation in several regions of the world has complicated the specific diagnosis of infections by these viruses in humans and other vertebrate hosts (10, 15). Cross-reactions in patients ultimately diagnosed with probable dengue virus infections have also been reported in evaluations of WNV testing assays (19).Previously we have reported the identification of WNVspecific neutralizing epitopes within structural domain III of the WNV envelope (E) protein (2). Earlier investigations with other flaviviruses have also reported the presence of virusspecific epitopes within this region of the E protein (e.g., see references 6, 22, and 25), and other authors have suggested the utility of domain III from dengue virus types 1 to 4 or JEV as antigens for specific serological diagnosis of infections with those flaviviruses (10, 24). These observations led us to investigate the utility of a recombinant, bacterially expressed domain III (r-EIII) antigen derived from the envelope protein of a North American WNV strain (385-99) for discrimination of WNV from other JE virus group infections. MATERIALS AND METHODSExpression and purification of recombinant WNV E protein domain III. Regions corresponding to structural domain III of the WNV strain 385-99 E protein were reverse transcription-PCR amplified for cloning and expression either as a glutathione S-transferase (GST) fusion using the pGEX-2T syste...

Enzyme-Linked Immunosorbent Assays Using Novel Japanese Encephalitis Virus Antigen Improve the Accuracy of Clinical Diagnosis of Flavivirus Infections

“…Strategies to differentiate current flavivirus infections have been proposed, such as by determining the IgMto-IgG ratio (15,36), by using recombinant EDIII or nonstructural protein 1 (NS1) as an antigen (2,36), or by epitopeblocking ELISA (12,19). All of these assays have limitations, including requiring the simultaneous testing of serum specimens for IgM and IgG, requiring paired acute-and convalescent-phase serum specimens, or the observation that not all infected individuals develop antibody against EDIII or NS1 antigen.…”

Monoclonal Antibody-Based Competitive Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying West Nile Virus-Neutralizing Antibodies in Horse Sera

“…There are several examples of the use of recombinant E proteins of WNV to develop diagnostic tests that are based on the indirect ELISA format (3,13,21,35). However, in a preliminary study using a competitive ELISA format, we found that WNV serum-neutralizing antibodies in most equine sera did not inhibit 5E8 binding to recombinant E protein expressed in Escherichia coli, perhaps due to conformational alterations in critical antigenic structures in the recombinant E protein.…”

“…Furthermore, the PRNT is not suitable for large-scale screening of susceptible animals, i.e., for monitoring population (or herd) immunity or measuring vaccine efficacy and infection. Recently, several enzyme-linked immunosorbent assays (ELISAs) have been developed and used in serologic testing for WNV infection, mainly in humans and horses (3,5,35). Although these ELISAs have been useful in detecting exposed individuals, test results do not directly correlate with the development of protective immunity against WNV in those individuals.…”

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

“…MIAFs raised against WNV lineage I, WNV lineage II, Japanese encephalitis virus (JEV) Nakayama, St. Louis encephalitis virus (SLEV) Parton, and Bagaza virus (BAGV) DakAr B209 were acquired from the WRCEVA collection. Previously described human sera from WNV-infected patients were also used (63). The deidentified sera were used under an exemption approved by the UTMB Institutional Review Board.…”