Most members of the genus Flavivirus (family Flaviviridae) are arthropod-borne viruses (arboviruses)transmitted by ticks or mosquitoes. Many of these viruses are significant agents of human and animaldisease. Some, such as the dengue viruses (DENV) and Japanese encephalitis virus (JEV) have long beenassociated with endemic and epidemic disease activity in tropical regions of the world and are a significantburden on public health resources of many developing countries. More recently, other flaviviruses haveemerged in new geographic regions and caused epidemics of human and/or animal disease, most notablythe introduction and subsequent spread of West Nile virus (WNV) in North America. In many cases,confirmatory diagnosis of infections with these agents requires specialized testing by reference laboratories.Serological diagnosis is also complicated by the extensive antigenic cross-reactivity that exists betweenflaviviruses. This cross-reactivity means that exposure to one virus can stimulate antibodies that will reactwith other flavivirus antigens in diagnostic assays. This is a particularly significant problem in areas wheremultiple flaviviruses circulate and cause endemic or epidemic disease. Previous studies by the applicanthave shown that a structural subunit of the envelope protein of WNV, designated domain III, containsepitopes recognized by primarily virus-specific antibodies and that use of a recombinant domain III proteinantigen in an ELISA assay provided dramatic improvements in specificity compared to ELISA usinginactivated whole virus antigen. Other investigators have reported similar observations for some otherflaviviruses. This improvement in specificity was comparable to the specificity obtained using neutralizationtesting, which is the current assay of choice for confirmation of flavivirus infection. Neutralization testing iscomplicated and requires working with live virus. Therefore, a simple immunoassay that can providecomparable or improved specificity, especially for the discrimination of the broadly cross-reactive immuneresponses, would offer significant advantages and potentially allow reliable surveillance and diagnosis ofHavivirus infections in the field or in standard clinical laboratories. The overall objective of this proposalis to develop candidate serological assays utilizing flavivirus E protein domain III antigens forsurveillance and diagnosis of flavivirus infections in situations where exposure to multiple virusescomplicates diagnosis using the current standard assays. To accomplish this goal, two specific aimsare proposed: 1. Demonstrate the diagnostic specificity of anti-E protein domain III antibodies in serumsamples obtained from experimentally inoculated mice in immunoassays using a panel of recombinantflavivirus domain III antigens representing virus types known or suspected to be transmitted in the Americas;2. Evaluate the reactivity of archived sera obtained from naturally infected and/or vaccinated humans inflavivirus-endemic areas in candidate immunoassays employing recombinant flavivirus domain III antigens.
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