Development of an enzyme-linked immunosorbent assay for Getah virus infection in horses using recombinant E2 protein as an antigen.
Abstract: Getah virus causes fever, skin eruptions, and limb edema in horses. For a high-throughput and time-saving method for serodiagnosis, we explored immunogenic antigens of Getah virus, and established an enzyme-linked immunosorbent assay (ELISA) using a recombinant protein. Western blot analysis using sera from infected horses showed strong reaction with viral antigens around 46 kDa corresponding to E1 or E2 glycoproteins. Recombinant E2 (rE2) protein reacted more strongly with infected horse sera than did rE1 protein in both Western blotting and ELISA. In ELISA using rE2 protein (rE2-ELISA), for all horses experimentally infected with Getah virus (n = 7), optical density (OD) exceeded the cutoff value at 14 days post-infection. ODs in five of nine vaccinated horses also slightly exceeded the cutoff value after vaccination. Among naturally infected horses (n = 28), 24 were seronegative in the acute sera, which turned seropositive in the convalescent sera. For the four horses seropositive in the acute sera, an endpoint method with serial dilutions of paired sera detected a ≥4-fold increase in titer. In conclusion, we established rE2-ELISA that could detect horse antibodies against Getah virus after experimental and natural infections; this should be useful in the diagnosis and surveillance of Getah virus infection.
Copyright © 2019 Elsevier B.V. All rights reserved.
Publication Date: 2019-06-15 PubMed ID: 31207276DOI: 10.1016/j.jviromet.2019.113681Google Scholar: Lookup
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Summary
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The research presents the formulation of an enzyme-linked immunosorbent assay (ELISA) to diagnose Getah virus infections in horses using a specific type of antigen, the recombinant E2 protein. The assay was found to be effective in identifying antibodies against the virus, suggesting its potential for use in the diagnosis and surveillance of Getah virus infection.
Research Methods
- The researchers analyzed various antigens of the Getah virus.
- Through Western blot analysis and the use of horse sera, they identified a strong reaction with viral antigens measuring around 46 kDa, which corresponded to E1 or E2 glycoproteins.
- E2 antigens showed a stronger reaction than the E1 antigens, both in Western blotting and ELISA.
Development and Validation of the ELISA
- The researchers created an ELISA using rE2 protein, which they referred to as rE2-ELISA.
- The efficacy of this assay was tested on seven horses that were experimentally infected with the Getah virus.
- They measured the optical density (OD) after 14 days post-infection, where all seven horses exceeded the cutoff value, demonstrating the ELISA’s ability to detect antibodies against the virus.
- In a subsequent test involving nine vaccinated horses, five outpaced the cutoff value.
Testing on Naturally Infected Horses
- In another validation effort, the researchers used rE2-ELISA on naturally infected horses.
- 24 out of 28 horses that initially tested negative (seronegative) in the initial stage (acute sera), later tested positive (seropositive) in the convalescence stage. This finding is significant because it demonstrates that the rE2 assay can successfully detect the seroconversion (the period of time during infection when antibodies develop and become detectable).
- The other four horses that were seropositive initially showed a significant increase in antibody titer, further supporting the validity of the rE2-ELISA.
Conclusion
- The rE2-ELISA, as developed and validated in this research, offers an efficient method to detect horse antibodies against the Getah virus after both experimental and natural infections.
- This suggests the potential of the rE2-ELISA for use in diagnosing and surveilling Getah virus infections in horses.
Cite This Article
APA
Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Kokado H.
(2019).
Development of an enzyme-linked immunosorbent assay for Getah virus infection in horses using recombinant E2 protein as an antigen.
J Virol Methods, 271, 113681.
https://doi.org/10.1016/j.jviromet.2019.113681 Publication
Researcher Affiliations
- Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan. Electronic address: hiroshi_bannai@jra.go.jp.
- Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan. Electronic address: manabu_nemoto@jra.go.jp.
- Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan. Electronic address: koji_tsujimura@jra.go.jp.
- Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan. Electronic address: takashi_yamanaka@jra.go.jp.
- Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi 329-0412, Japan. Electronic address: hiroshi_kokado@jra.go.jp.
MeSH Terms
- Alphavirus / immunology
- Alphavirus / isolation & purification
- Alphavirus Infections / blood
- Alphavirus Infections / diagnosis
- Alphavirus Infections / veterinary
- Animals
- Antibodies, Viral / blood
- Antigens, Viral / immunology
- Enzyme-Linked Immunosorbent Assay / veterinary
- Horse Diseases / blood
- Horse Diseases / diagnosis
- Horses / virology
- Recombinant Proteins / immunology
- Viral Envelope Proteins / genetics
- Viral Envelope Proteins / immunology
Citations
This article has been cited 6 times.- Qiu X, Cao X, Shi N, Zhang H, Zhu X, Gao Y, Mai Z, Jin N, Lu H. Development and application of an indirect ELISA for detecting equine IgG antibodies against Getah virus with recombinant E2 domain protein. Front Microbiol 2022;13:1029444.
- Sun Q, Xie Y, Guan Z, Zhang Y, Li Y, Yang Y, Zhang J, Li Z, Qiu Y, Li B, Liu K, Shao D, Wang J, Ma Z, Wei J, Li P. Seroprevalence of Getah virus in Pigs in Eastern China Determined with a Recombinant E2 Protein-Based Indirect ELISA. Viruses 2022 Sep 30;14(10).
- Shi N, Qiu X, Cao X, Mai Z, Zhu X, Li N, Zhang H, Zhang J, Li Z, Shaya N, Lu H, Jin N. Molecular and serological surveillance of Getah virus in the Xinjiang Uygur Autonomous Region, China, 2017-2020. Virol Sin 2022 Apr;37(2):229-237.
- Yuan Y, Hao Y, Peng C, Zhang D, Ma W, Xiao P, Li N. From transmission to adaptive evolution: genomic surveillance of Getah virus. Front Cell Infect Microbiol 2025;15:1513392.
- Lan J, Duan L, Liu X, Zhou Y, Zeng B, Chen S, Ye Y, Huang D, Wan G, Zhang F, Song D. Seroprevalence of Getah virus in pigs in Southeast China determined with a recombinant Cap protein-based indirect ELISA. Front Microbiol 2025;16:1547670.
- Zhong D, Zheng J, Ma Z, Wang Y, Wei J. Rapid Detection of Getah Virus Antibodies in Horses Using a Recombinant E2 Protein-Based Immunochromatographic Strip. Animals (Basel) 2024 Aug 8;14(16).
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