Serological differentiation of West Nile, Usutu, and tick-borne encephalitis virus antibodies in birds and horses using mutant E protein ELISAs.
Abstract: 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.
© 2025. The Author(s).
Publication Date: 2025-08-06 PubMed ID: 40770485PubMed Central: PMC12328586DOI: 10.1038/s41598-025-14448-4Google Scholar: Lookup
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Summary
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This research presents a method to differentiate between antibodies of West Nile virus, Usutu virus, and tick-borne encephalitis virus in ducks, geese, chickens, and horses. This reliable differentiation is realized with high sensitivity and specificity using enzyme-linked immunosorbent assays, without the need for virus neutralization tests, providing improved efficiency for veterinary diagnosis and seroprevalence studies.
Overview of Research Topic
- The research primarily focuses on West Nile virus (WNV), Usutu virus (USUV), and tick-borne encephalitis virus (TBEV), all of which are zoonotic orthoflaviviruses that are endemic worldwide and often co-circulate in the same areas.
- Studies of these viruses are vital to monitoring their spread and understanding the potential infection risks for humans, mainly through carriers like birds and horses.
- A significant problem in serological studies is the cross-reactivity of antibodies to these flaviviruses, which can cause difficulties in differentiation during enzyme-linked immunosorbent assays (ELISAs), leading to the need for laborious virus neutralization tests (VNTs) in high biosafety level laboratories.
Objective and Methodology of the Study
- The research aimed to present an ELISA method, using recombinant flavivirus E proteins with point mutations in the conserved fusion loop domain (known as Equad proteins), which can differentiate between IgY or IgG antibodies against the three aforementioned viruses in ducks, geese, chickens, and horses.
- A total of 169 duck and goose sera, 101 chicken sera, and 136 horse sera were tested using these Equad ELISAs.
Findings and Implications
- The research found high sensitivity and specificity in the differentiation of WNV, USUV, and TBEV specific antibodies using Equad ELISAs, even more so when a pre-absorption step was included for the differentiation of WNV and USUV antibodies.
- This method enables reliable differentiation without the need for virus neutralization tests, making it more efficient and less demanding on laboratory resources.
- Consequently, this method of differentiation using Equad ELISAs has significant implications for the conduct of seroprevalence studies and routine veterinary diagnosis in relation to WNV, USUV, and TBEV.
Cite This Article
APA
Schwarzer A, Ziegler U, Fertey J, Kreuz M, Vahlenkamp TW, Groschup MH, Ulbert S.
(2025).
Serological differentiation of West Nile, Usutu, and tick-borne encephalitis virus antibodies in birds and horses using mutant E protein ELISAs.
Sci Rep, 15(1), 28752.
https://doi.org/10.1038/s41598-025-14448-4 Publication
Researcher Affiliations
- Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler Institut, Südufer 10, 17493, Greifswald-Insel Riems, Germany.
- Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler Institut, Südufer 10, 17493, Greifswald-Insel Riems, Germany.
- Fraunhofer Institute for Cell Therapy and Immunology, Department of Infection Research and Diagnostics, Perlickstraße 1, 04103, Leipzig, Germany.
- Fraunhofer Institute for Cell Therapy and Immunology, Department of Medical Bioinformatics,, Perlickstraße 1, 04103, Leipzig, Germany.
- Institute of Virology, Faculty of Veterinary Medicine, Leipzig University, An den Tierkliniken 29, 04103, Leipzig, Germany.
- Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler Institut, Südufer 10, 17493, Greifswald-Insel Riems, Germany.
- Fraunhofer Institute for Cell Therapy and Immunology, Department of Infection Research and Diagnostics, Perlickstraße 1, 04103, Leipzig, Germany. sebastian.ulbert@izi.fraunhofer.de.
MeSH Terms
- Animals
- Horses / virology
- Antibodies, Viral / blood
- Antibodies, Viral / immunology
- West Nile virus / immunology
- Enzyme-Linked Immunosorbent Assay / methods
- Flavivirus / immunology
- Encephalitis Viruses, Tick-Borne / immunology
- Birds / virology
- Flavivirus Infections / veterinary
- Flavivirus Infections / immunology
- Flavivirus Infections / virology
- Flavivirus Infections / diagnosis
- West Nile Fever / veterinary
- West Nile Fever / immunology
- West Nile Fever / virology
- Encephalitis, Tick-Borne / immunology
- Encephalitis, Tick-Borne / veterinary
- Encephalitis, Tick-Borne / blood
- Encephalitis, Tick-Borne / virology
- Encephalitis, Tick-Borne / diagnosis
- Viral Envelope Proteins / immunology
- Viral Envelope Proteins / genetics
- Immunoglobulin G / blood
- Immunoglobulin G / immunology
- Chickens
Conflict of Interest Statement
Declarations. Competing interests: S.U. is on a patent application that includes the proteins described in this study. This does not alter his adherence to policies on sharing data and materials. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. A.S., U.Z., J.F., M.K., T.W.V. and M.H.G. declare no potential conflict of interest. Ethics approval: Duck, goose and chicken sera were used according to the ethical approvals by the administration of the federal state of Mecklenburg-Western Pomerania, Germany (LALLF reference numbers 7221.3-2-042/17, 7221.3-2-003/23, 7221.3-1-075/16, 7221.3-1.1-018/18 and 7221.3-1-031/20) and in accordance with the relevant guidelines. All methods are reported in accordance with ARRIVE guidelines. Blood samples from horses were collected during routine diagnostic testing by veterinarians for which, according to German law, ethical approval was not required.
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