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. 2003 Sep;41(9):4217-23.
doi: 10.1128/JCM.41.9.4217-4223.2003.

Immunoassay targeting nonstructural protein 5 to differentiate West Nile virus infection from dengue and St. Louis encephalitis virus infections and from flavivirus vaccination

Susan J Wong  1 , Rebekah H Boyle, Valerie L Demarest, Anh N Woodmansee, Laura D Kramer, Hongmin Li, Michael Drebot, Raymond A Koski, Erol Fikrig, Denise A Martin, Pei-Yong Shi
Affiliations

Immunoassay targeting nonstructural protein 5 to differentiate West Nile virus infection from dengue and St. Louis encephalitis virus infections and from flavivirus vaccination

Susan J Wong et al. J Clin Microbiol. 2003 Sep.

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.

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Figures

FIG. 1.
FIG. 1.
(A) WNV genome structure. The recombinant proteins used in this study are shaded. (B and C) Purified NTPase/helicase domain of NS3 and full-length NS5 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gels were stained with Coomassie blue. (D) ATPase activity of the recombinant NTPase/helicase domain of WNV NS3. In the presence of recombinant NS3, [α-32P]ATP was hydrolyzed to [α-32P]ADP and phosphate (lane 2). No ATP is hydrolyzed in the absence of NS3 (lane 1). (E) RDRP activity of the recombinant NS5. The RDRP activity of NS5 was assayed with a WNV subgenomic RNA transcript (890 nt) containing a large deletion from nucleotide 269 to 10408. The reaction products (RXT) were labeled with [α-32P]UTP, and the products of 1× and 2X forms of RNA were analyzed on a denaturing polyacrylamide gel followed by autoradiography (lane 1). A 32P-labeled template RNA was loaded as a size control (lane 2).
FIG. 2.
FIG. 2.
MIAs using recombinant WNV NS5 (A), NS3 (B), and E protein (C). The MFI of each WNV patient serum is plotted against days after symptom onset. Dashed lines indicate assay cutoff levels. X, samples not tested. (D) Time course of reactivity to NS5 and E protein for sera from a patient infected with WNV. MFIs from NS5- and E-protein-based assays are indicated by solid and dashed lines, respectively. The cutoff values of the assays are indicated by corresponding horizontal lines.
FIG. 2.
FIG. 2.
MIAs using recombinant WNV NS5 (A), NS3 (B), and E protein (C). The MFI of each WNV patient serum is plotted against days after symptom onset. Dashed lines indicate assay cutoff levels. X, samples not tested. (D) Time course of reactivity to NS5 and E protein for sera from a patient infected with WNV. MFIs from NS5- and E-protein-based assays are indicated by solid and dashed lines, respectively. The cutoff values of the assays are indicated by corresponding horizontal lines.
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