Table 2: Antigen formulations to improve serological assay specificity.

AntigenReferenceAssay FormatSerumSensitivitySpecificity

E protein: removal of DII cross-reactive epitopes from virus-like particles. One antigen for WNV and one antigen for SLEVRoberson et al., 2007 [136]IgM ELISAHuman. 134 sera including those with disease states classified as SLEV, WNV, JEV or DENVMutant WNV antigen: 100%*
Mutant SLEV antigen: 100%#
Mutant WNV antigen: 88.6%* versus 70.9% for WT§
Mutant SLEV antigen: 88.7%# versus 69.1% for WT§

WNV E protein subunit (r-EIII)Beasley et al., 2004 [137]WNV ELISAPolyclonal mouse immune ascitic fluid- anti-WNV, -JEV, -SLEV, -MVEV, -DENV, -YFVStrong reactivity of anti-WNV ascitic fluid with r-EIII at 1 : 64 dilutionNegligible reactivity of anti-JEV, -SLEV, -MVEV, -DENV and -YFV ascitic fluid with r-EDIII at 1 : 64 dilution.
IgG ELISAHuman: PRNT/HI confirmed WNV pos ( 𝑛 = 1 1 ); WNV neg ( 𝑛 = 4 , plus 1 SLEV pos) 100% 100%
IgG ELISA Horse: 57 PRNT/HI confirmed 89%( 𝑛 = 3 5 ) 87.5% ( 𝑛 = 1 6 )

WNV E protein peptide DIII (Ep15)Herrmann et al., 2007 [138]IgG ELISAHuman. 66 sera including 7 WNV pos, 3 WN neg, 2 DENV pos, 54 unknown.67%100%

WNV E protein peptide DI (WN19)Hobson-Peters et al., 2008, 2011 [60, 61]Western BlotHorse. VNT confirmed69% ( 𝑛 = 1 1 WNV pos)[60]100% ( 𝑛 = 4 WNV/MVEV neg)
80% WNV PRNT confirmed field samples ( 𝑛 = 5 ) [61]Cannot differentiate MVEV-positive sera. 100% MVEV positive sera ( 𝑛 = 4 ) bound WN19.

JEV and DENV prM-native viral antigen Cardosa et al., 2002 [89]Western BlotHuman. 16 JEV pos, 22 DENV pos.DENV assay: 95%
JEV assay: 100%
DENV assay: 100%
JEV assay: 73%
Pig. 31 JEV posJEV assay: 93.5%DENV assay: 100%

WNV prM-native viral antigenSetoh et al., 2011 [62]Western BlotHorse. VNT confirmed. 16 WNV pos, 6 WNV negative (2 of these Kokobera positive)87.5%100%

JEV prM/M peptideHua et al., 2010 [139]ELISARabbitPeptide bound by anti-JEV serumPeptide not bound by anti-WNV and -DENV sera.

*Ability to distinguish WNV infections from other arbovirus infections.
§WT = Wild Type.
#Ability to distinguish SLEV infections from other arbovirus infections.
After removal of 6 strong IgM positives and classifying equivocal results as false negative (for sensitivity) or false positive (for specificity).
Upon comparison with commercial IgG kit.
Percentages based on those serum samples that did not cross-react with the scFv carrier protein.