Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination

Abstract

West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.West Nile virus (WNV) is a member of the genus Flavivirus, which includes many significant human pathogens of global epidemiological importance, including four serotypes of dengue (DEN) virus, yellow fever (YF) virus, Japanese encephalitis (JE) virus, St. Louis encephalitis (SLE) virus, and tickborne encephalitis (TBE) virus, as well as WNV (3). Among them, DEN virus, YF virus, TBE virus, JE virus, and WNV are listed by the National Institutes of Health as potential biodefense pathogens. Since its introduction into the United States in 1999, WNV has resulted in more than 4,156 known human cases, with 284 deaths (for updates, see http://www.cdc.gov /ncidod/dvbid/westnile/surv&controlCaseCount03.htm). Recent studies have shown that, besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion, organ transplantation (9), breast feeding (8), intrauterine exposure (6), and laboratory-acquired infection (7). These findings have underlined the importance of developing an accurate serologic assay for diagnosis of WNV infection.Flavivirus genomic RNA contains a single open reading frame encoding 10 viral proteins: three structural and seven nonstructural (NS) proteins (Fig. 1A). Viral envelope protein (E protein) (14), NS1 (18,25,26), and NS3 (24) are the most immunogenic proteins during flavivirus infection (15). The current serologic diagnosis of WNV infection is based on detection of antibodies against viral structural proteins, mainly the E protein (12, 21). Unfortunately, the high cross-reactivity of the E protein among flaviviruses limits the specificity of the assay. Positive sera or spinal fluids identified by the current assay must be verified by cross-species plaque reduction neutralization tests (PRNT) to exclude the possibility of infection with cross-reactive viruses such as SLE and DEN. These confirmatory tests have to be performed in level 3 biocontainment for many flaviviruses and substanti...

Dengue Virus Seroconversion in Travelers to Dengue-Endemic Areas

“…[12][13][14][15] Many travelers to dengue-endemic areas receive a vaccine for YF or JE for travel to locations where they could encounter one of these flavivirus infections. Our PRNT results demonstrated false-positive anti-DENV IgM ELISA results due to crossreactivity between flaviviruses.…”

“…Given the potential cross-reactions of dengue with other arboviral infections when tested by ELISA for anti-DENV IgM result, these samples were tested for DENV and YF by plaque-reduction neutralization test (PRNT) at the CDC Arboviral Diseases Branch according to a protocol based on WHO guidelines; PRNT for JE was planned but could not be performed. 12 Funding limited number of samples that could be confirmed by PRNT; we were not able to test samples of subjects with seroconversion of anti-DENV IgG who received JE or YF vaccines.…”

Dengue: a continuing global threat

“…In general, an IgG ELISA lacks specificity within the flavivirus serocomplex groups, however it has been demonstrated that the IgG response to the prM membrane glycoprotein is specific to individual flaviviruses as no crossreactivity was observed in sera collected from individuals infected with dengue or Japanese encephalitis virus 85 . Similarly, it has been demonstrated that IgG specific for the NS5 protein can potentially discriminate between infections caused by West Nile, dengue and St Louis encephalitis viruses 86 . Finally, dengue-specific IgG was shown to have high specificity in an assay using a recombinant polypeptide located in the N-terminal region of the envelope protein 87 .…”

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

“…The fact that NS5 is a reliable serological marker of WNV infection (Wong et al, 2003) suggests that further investigation of immunodominance of the epitopes identified here during human and animal infections is warranted.…”