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Virology journal2025; 22(1); 276; doi: 10.1186/s12985-025-02898-1

Development of an African horse sickness VP6 DIVA diagnostic ELISA.

Abstract: African horse sickness (AHS) is a severe, noncontagious disease of equines caused by the African horse sickness virus (AHSV). The virus has nine serotypes and is transmitted by the midge. AHS is endemic in South Africa and other sub-Saharan African countries. Currently, the disease is managed using a live attenuated vaccine manufactured by Onderstepoort Biological Products (OBP). Although this vaccine has been in use for decades, it has several drawbacks, including the possibility of reversion to virulence, and it does not allow for the differentiation of infected horses from vaccinated horses (DIVA). Previously, our group developed recombinant AHSV serotype 4 and 5 virus-like particle (VLP) vaccine candidates in plants that elicited an immune response in guinea pigs and horses. In this research, we aimed to develop a diagnostic enzyme-linked immunosorbent assay (ELISA) using an AHSV-VP6 antigen expressed in plants, allowing for the differentiation of horses infected with the virus from those vaccinated with VLP vaccine candidates. For this DIVA ELISA, we utilized a robust, cost-effective, and easily scalable manufacturing process that employs transient expression of VP6 in . AHSV-VP6 sequences from all nine serotypes were aligned to obtain a consensus sequence, which was then used to design the gene. The gene was successfully expressed in plants via -mediated infiltration. The VP6 protein was extracted from infiltrated leaves and purified. A purified yield of approximately 7.7 mg of recombinant VP6/kg fresh weight leaf material was obtained. The VP6 protein was also expressed in , yielding a purified product of 9.4 mg/L. Preliminary data revealed that AHSV-VP6 antigen expressed in both plants and could be used to differentiate between sera from infected horses and those vaccinated with the candidate AHSV4 and AHSV5 VLP vaccines. The plant-produced VP6 could detect more anti-VP6 antibodies than the produced VP6. In this study, we expressed the AHSV-VP6 protein in plants, which enabled the differentiation of infected AHSV horse sera from those of horses vaccinated with the candidate VLP vaccines. To our knowledge, this is the first evidence of AHSV-VP6 expression in plants and the first demonstration of its diagnostic ability. The online version contains supplementary material available at 10.1186/s12985-025-02898-1.
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Publication Date: 2025-08-12 PubMed ID: 40796889PubMed Central: PMC12344834DOI: 10.1186/s12985-025-02898-1Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study focused on developing a diagnostic test to distinguish horses infected with African horse sickness virus (AHSV) from those vaccinated with a new candidate vaccine, using a plant-produced version of a viral protein called VP6.
  • The researchers produced the VP6 protein in plants and another expression system, created an ELISA test, and demonstrated its ability to differentiate infected from vaccinated horses.

Background on African Horse Sickness and Current Challenges

  • African horse sickness (AHS) is a severe, noncontagious disease affecting horses caused by the African horse sickness virus (AHSV), which has nine different serotypes.
  • The virus is transmitted by midges and is widespread in South Africa and other sub-Saharan African countries.
  • Current vaccination relies on a live attenuated vaccine produced by Onderstepoort Biological Products (OBP), which has been used for many years.
  • However, this live vaccine has drawbacks:
    • Potential to revert to a virulent form, endangering horses.
    • It does not enable differentiation between horses that are naturally infected and those that were vaccinated—a challenge known as the DIVA (Differentiating Infected from Vaccinated Animals) problem.

Previous Research and Current Study Goals

  • The authors’ group previously developed vaccine candidates based on virus-like particles (VLPs) of AHSV serotypes 4 and 5 produced in plants.
  • These VLP vaccines triggered immune responses in guinea pigs and horses, making them promising alternatives.
  • The current study aimed to develop a diagnostic ELISA test using the VP6 protein of AHSV to address the DIVA challenge specifically for horses vaccinated with the VLP vaccine candidates.
  • The VP6 protein was selected because it is a viral antigen that can potentially distinguish natural infection from vaccination responses.

Methodology: Production of VP6 Protein

  • The researchers started by aligning VP6 gene sequences from all nine known AHSV serotypes to create a consensus gene, which would be applicable to all serotypes.
  • This consensus VP6 gene was then engineered for expression in plants and another system called Escherichia coli (E. coli).
  • Plant Expression:
    • Used Nicotiana benthamiana plants, a common model for transient protein expression via Agrobacterium-mediated infiltration.
    • VP6 protein was successfully produced in the plant leaves.
    • Purification yielded approximately 7.7 mg of recombinant VP6 per kilogram of fresh leaf weight.
  • E. coli Expression:
    • VP6 protein was also expressed and purified from bacterial cultures.
    • Yield was about 9.4 mg of purified VP6 protein per liter of culture.

Development and Use of DIVA ELISA

  • The purified VP6 proteins from both expression systems were used as antigens in ELISA tests to detect anti-VP6 antibodies in horse sera.
  • They tested sera from:
    • Horses naturally infected with AHSV.
    • Horses vaccinated with the candidate VLP vaccines for serotypes 4 and 5.
  • Results showed that the VP6 antigen could differentiate infected horses, which produce anti-VP6 antibodies, from vaccinated horses that do not produce these antibodies because the VLP vaccines lack VP6.
  • The plant-produced VP6 antigen showed higher sensitivity in detecting anti-VP6 antibodies compared to the E. coli-produced VP6, indicating the plant system might produce a more immunologically relevant protein.

Significance and Novelty of the Study

  • This is the first report of expressing the AHSV VP6 protein in plants.
  • The study demonstrates for the first time that plant-produced VP6 can be used as an antigen in a diagnostic ELISA for DIVA purposes related to African horse sickness.
  • The development of this DIVA diagnostic tool is important because:
    • It allows differentiation between infected and vaccinated animals, which is essential for disease surveillance and outbreak control.
    • It complements newer, safer VLP-based vaccines that lack the VP6 protein, increasing vaccine safety and diagnostic capability.
    • The plant-based process is cost-effective and scalable, suitable for broader implementation in endemic regions.

Additional Resources

  • The paper includes supplementary material that can be accessed online, providing more detailed information and data supporting the study’s conclusions.

Cite This Article

APA
Tinarwo M, Dennis SJ, Hitzeroth II, Meyers AE, Rybicki EP, Mbewana S. (2025). Development of an African horse sickness VP6 DIVA diagnostic ELISA. Virol J, 22(1), 276. https://doi.org/10.1186/s12985-025-02898-1

Publication

Virology journal
ISSN: 1743-422X
NlmUniqueID: 101231645
Country: England
Language: English
Volume: 22
Issue: 1
Pages: 276
PII: 276

Researcher Affiliations

Tinarwo, Munyaradzi
  • Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, 7700, South Africa.
Dennis, Susan J
  • Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, 7700, South Africa.
Hitzeroth, Inga I
  • Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, 7700, South Africa.
Meyers, Ann E
  • Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, 7700, South Africa.
Rybicki, Edward P
  • Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, 7700, South Africa.
  • Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa.
Mbewana, Sandiswa
  • Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, 7700, South Africa. s.mbewana@uct.ac.za.

Conflict of Interest Statement

Declarations. Ethics approval and consent to participate: This work was done at the BRU-UCT and was covered by appropriate ethics approvals granted by the Faculty of Health Sciences Animal Ethics Committee, University of Cape Town. FHS AEC REF NO: 020_025. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

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