Schwarzer et al.
Serological differentiation of West Nile, Usutu, and tick-borne encephalitis virus antibodies in birds and horses using mutant E protein ELISAs. Scientific Reports. 2025;15:28752. doi:10.1038/s41598-025-14448-4.
Specific serological diagnosis of flavivirus infections is challenging due to the strong antigenic similarity among closely related flaviviruses. This similarity leads to highly cross-reactive antibody responses, particularly in ELISA systems based on the flaviviral envelope glycoprotein E.
To address this, a novel ELISA has been developed in which specific mutations were introduced into the fusion loop (FL) of the envelope glycoprotein E (gE). These mutations enable reliable differentiation of West Nile virus, Usutu virus, and tick-borne encephalitis (TBE) virus infections in horses and birds. For assay development, the ectodomains of gE for each of the three viruses were engineered to contain four-point mutations near the FL domain and were expressed in a Drosophila S2 cell system.
This newly established ELISA has the potential to replace the virus neutralization test, which is currently considered the gold standard but requires biosafety level 3 facilities. Using the modified ELISA, the distribution of co-circulating flaviviruses in parts of Germany was analyzed in both avian (ducks, geese, chickens) and equine sera.
By incorporating a modified FL domain, the assay effectively minimized cross-reactive binding among closely related West Nile, Usutu, and TBE virus IgY and IgG antibodies. The test system demonstrated high specificity and sensitivity, while being rapid, cost-effective, and sustainable. Importantly, it allows for reliable and efficient serological diagnosis of flavivirus infections in standard diagnostic laboratories and for use in seroprevalence studies.