Serological differentiation of West Nile, Usutu, and tick-borne encephalitis virus antibodies in birds and horses using mutant E protein ELISAs.

West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) are worldwide endemic zoonotic orthoflaviviruses, often co-circulating in the same areas. Serological studies in animals, mostly birds and horses, are important means to monitor the spread of these viruses and the infection risks for humans. However, cross-reactive antibodies to these structurally similar flaviviruses frequently impact serological differentiation in enzyme-linked immunosorbent assays (ELISAs), hence time-consuming virus neutralization tests (VNTs) have to be employed in laboratories with high biosafety level. This study presents ELISAs using recombinant flavivirus E proteins with point mutations in the conserved fusion loop domain (Equad proteins) for differentiating IgY or IgG antibodies against WNV, USUV or TBEV in ducks, geese, chickens and horses. Panels of 169 duck and goose sera, 101 chicken sera and 136 horse sera were tested in Equad ELISAs, which resulted in high sensitivity and specificity, further improved by a pre-absorption step for the differentiation of WNV and USUV antibodies. Equad ELISAs for poultry and horse sera enable the reliable differentiation of WNV, USUV and TBEV specific antibodies without the need for VNTs, which has important implications for conducting seroprevalence studies as well as for veterinary routine diagnosis.