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Home / Equine Research Bank / Virol J / Development of an African horse sickness VP6 DIVA diagnostic...
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.

References

This article includes 55 references
  1. OIE. African horse sickness (Infection with African horse sickness virus) 2019 [Available from: https://www.oie.int/standard-setting/terrestrial-manual/access-online
  2. Mellor PS, Hamblin C. African horse sickness. Vet Res 2004;35(4):445–66.
    pubmed: 15236676
  3. Zientara S, Weyer CT, Lecollinet S. African horse sickness. Sci Tech Rev Office Int Des Epizooties (Paris) 2015;34(2):315–27.
    pubmed: 26601437
  4. Dennis SJ, Meyers AE, Hitzeroth II, Rybicki EP. African horse sickness: A review of current Understanding and vaccine development. Viruses 2019;11(9).
    pmc: PMC6783979pubmed: 31514299
  5. Abul Rahman S, Reed K. The management and welfare of working animals: identifying problems, seeking solutions and anticipating the future. Rev Sci Tech 2014;33(1):197–202.
    pubmed: 25000792
  6. Beck AM, Meyers NM. Health enhancement and companion animal ownership. Annu Rev Public Health 1996;17:247–57.
    pubmed: 8724226
  7. Suggett RH. Horses and the rural economy in the united Kingdom. Equine Vet J Suppl 1999(28):31–7.
    pubmed: 11314232
  8. Carpenter S, Mellor PS, Fall AG, Garros C, Venter GJ. African horse sickness virus: history, transmission, and current status. Ann Rev Entomol 2017;62:343–58.
    pubmed: 28141961
  9. Bremer CW, Huismans H, Van Dijk AA. Characterization and cloning of the African horsesickness virus genome. J Gen Virol 1990;71:793–9.
    pubmed: 2324709
  10. de Vos CJ, Hoek CA, Nodelijk G. Risk of introducing African horse sickness virus into the Netherlands by international equine movements. Prev Vet Med 2012;106(2):108–22.
    pubmed: 22341773
  11. Bunpapong N, Charoenkul K, Nasamran C, Chamsai E, Udom K, Boonyapisitsopa S. African horse sickness virus serotype 1 on horse farm, thailand, 2020. Emerg Infect Dis 2021;27(8):2208–11.
    pmc: PMC8314833pubmed: 34287126
  12. King S, Rajko-Nenow P, Ashby M, Frost L, Carpenter S, Batten C. Outbreak of African horse sickness in thailand, 2020. Transbound Emerg Dis 2020;67(5):1764–7.
    pubmed: 32593205
  13. Grewar JD, Kotze JL, Parker BJ, van Helden LS, Weyer CT. An entry risk assessment of African horse sickness virus into the controlled area of South Africa through the legal movement of equids. PLoS ONE 2021;16(5):e0252117.
    pmc: PMC8153453pubmed: 34038466
  14. Weyer CT, Grewar JD, Burger P, Rossouw E, Lourens C, Joone C. African horse sickness caused by genome reassortment and reversion to virulence of live, attenuated vaccine viruses, South africa, 2004–2014. Emerg Infect Dis 2016;22(12):2087–96.
    pmc: PMC5189153pubmed: 27442883
  15. Roy P, Bishop DH, Howard S, Aitchison H, Erasmus B. Recombinant baculovirus-synthesized African horsesickness virus (AHSV) outer-capsid protein VP2 provides protection against virulent AHSV challenge. J Gen Virol 1996;77(Pt 9):2053–7.
    pubmed: 8811002
  16. Kanai Y, van Rijn PA, Maris-Veldhuis M, Kaname Y, Athmaram TN, Roy P. Immunogenicity of Recombinant VP2 proteins of all nine serotypes of African horse sickness virus. Vaccine 2014;32(39):4932–7.
    pmc: PMC4148702pubmed: 25045805
  17. Aksular M, Calvo-Pinilla E, Marin-Lopez A, Ortego J, Chambers AC, King LA. A single dose of African horse sickness virus (AHSV) VP2 based vaccines provides complete clinical protection in a mouse model. Vaccine 2018;36(46):7003–10.
    pmc: PMC6219453pubmed: 30309744
  18. O’Kennedy MM, Roth R, Ebersohn K, du Plessis LH, Mamputha S, Rutkowska DA. Immunogenic profile of a plant-produced nonavalent African horse sickness viral protein 2 (VP2) vaccine in IFNAR-/- mice. PLoS ONE 2024;19(4):e0301340.
    pmc: PMC11020708pubmed: 38625924
  19. Romito M, Du Plessis Dh Fau -, Viljoen GJ, Viljoen GJ. Immune responses in a horse inoculated with the VP2 gene of African horsesickness virus. Onderstepoort J Veterinaty Rep 1999;66(2):0030–2465.
    pubmed: 10486832
  20. Stone-Marschat MA, Moss SR, Burrage TG, Barber ML, Roy P, Laegreid WW. Immunization with VP2 is sufficient for protection against lethal challenge with African horsesickness virus type 4. Virology 1996;220(1):219–22.
    pubmed: 8659117
  21. Guthrie AJ, Quan M, Lourens CW, Audonnet JC, Minke JM, Yao J. Protective immunization of horses with a Recombinant Canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus. Vaccine 2009;27(33):4434–8.
    pubmed: 19490959
  22. Calvo-Pinilla E, de la Poza F, Gubbins S, Mertens PP, Ortego J, Castillo-Olivares J. Vaccination of mice with a modified vaccinia Ankara (MVA) virus expressing the African horse sickness virus (AHSV) capsid protein VP2 induces virus neutralising antibodies that confer protection against AHSV upon passive immunisation. Virus Res 2014;180:23–30.
    pubmed: 24333835
  23. van de Water SG, van Gennip RG, Potgieter CA, Wright IM, van Rijn PA. VP2 exchange and NS3/NS3a deletion in African horse sickness virus (AHSV) in development of disabled infectious single animal vaccine candidates for AHSV. J Virol 2015;89(17):8764–72.
    pmc: PMC4524073pubmed: 26063433
  24. Lulla V, Losada A, Lecollinet S, Kerviel A, Lilin T, Sailleau C. Protective efficacy of multivalent replication-abortive vaccine strains in horses against African horse sickness virus challenge. Vaccine 2017;35(33):4262–9.
    pmc: PMC5518735pubmed: 28625521
  25. van Rijn PA, Maris-Veldhuis MA, Potgieter CA, van Gennip RGP. African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS disabled infectious single animal (DISA) vaccine platform. Vaccine 2018;36(15):1925–33.
    pubmed: 29525278
  26. Wade-Evans AM, Woolhouse T, O’Hara R, Hamblin C. The use of African horse sickness virus VP7 antigen synthesised in bacteria and anti-VP7 monoclonal antibodies in a competitive ELISA. J Virol Methods 1993;45:179–88.
    pubmed: 8113344
  27. du Plessis M, Cloete M, Fau - Aitchison H, Aitchison H, Van Fau - AA, Van Dijk AA. Protein aggregation complicates the development of baculovirus-expressed African horsesickness virus serotype 5 VP2 subunit vaccines. Onderstepoort J Veterinary Res 1998;65(4):0030–2465.
    pubmed: 10192846
  28. Maree S, Maree FF, Putterill JF, de Beer TAP, Huismans H, Theron J. Synthesis of empty African horse sickness virus particles. Virus Res 2016;213:184–94.
    pubmed: 26686484
  29. Dennis SJ, Meyers AE, Guthrie AJ, Hitzeroth II, Rybicki EP. Immunogenicity of plant-produced African horse sickness virus-like particles: implications for a novel vaccine. Plant Biotechnol J 2018;16(2):442–50.
    pmc: PMC5787833pubmed: 28650085
  30. Dennis SJ, O’Kennedy MM, Rutkowska D, Tsekoa T, Lourens CW, Hitzeroth II. Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses. Vet Res 2018;49(1):105.
    pmc: PMC6389048pubmed: 30309390
  31. Rutkowska DA, Mokoena NB, Tsekoa TL, Dibakwane VS, O’Kennedy MM. Plant-produced chimeric virus-like particles - a new generation vaccine against African horse sickness. BMC Vet Res 2019;15(1):432.
    pmc: PMC6892175pubmed: 31796116
  32. Dennis SJ. Development of plant-produced African horse sickness virus vaccines. 2019.
  33. Chuma T, Le Blois H, Sanchez-Vizcaino JM, Diaz-Laviada M, Roy P. Expression of the major core antigen VP7 of African horsesickness virus by a Recombinant baculovirus and its use as a group-specific diagnostic reagent. J Gen Virol 1992;73(Pt 4):925–31.
    pubmed: 1378881
  34. Oladosu LA, Olayeye OD, Baba SS, Omilabu SA. Isolation and identification of African horse sickness virus during an outbreak in Lagos. Nigeria Rev Sci Tech 1993;12(3):873–7.
    pubmed: 8219337
  35. Tessler J. Detection of African horsesickness viral antigens in tissues by Immunofluorescence. Can J Comp Med 1972;36(2).
    pmc: PMC1319637pubmed: 4259931
  36. van Schalkwyk A, Ferreira ML, Romito M. Using a new serotype-specific polymerase chain reaction (PCR) and sequencing to differentiate between field and vaccine-derived African horse sickness viruses submitted in 2016/2017. J Virol Methods 2019;266:89–94.
    pubmed: 30721715
  37. Wang Y, Ong J, Ng OW, Songkasupa T, Koh EY, Wong JPS. Development of differentiating infected from vaccinated animals (DIVA) Real-Time PCR for African horse sickness virus serotype 1. Emerg Infect Dis 2022;28(12):2446–54.
    pmc: PMC9707579pubmed: 36417933
  38. Laegreid WW. Diagnosis of African horse sickness. Comp Immunol Microbiol Infect Dis 1994;17(3):297–303.
    pubmed: 8001351
  39. Maree S, Paweska JT. Preparation of Recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-specific IgG antibodies in horse Sera. J Virol Methods 2005;125(1):55–65.
    pubmed: 15737417
  40. Hamblin C, Graham SD, Anderson EC, Crowther JR. A competitive ELISA for the detection of group-specific antibodies to African horse sickness virus. Epidemiol Infect 1990;104:303–12.
    pmc: PMC2271754pubmed: 2108871
  41. Rubio C, Cubillo MA, Hooghuis H, Sanchez-Vizcaino JM, Diaz-Laviada M, Plateau E. Validation of ELISA for the detection of African horse sickness virus antigens and antibodies. 1998.
    pubmed: 9785516
  42. Turnbull PJ, Cormack SB, Huismans H. Characterization of the gene encoding core protein VP6 of two African horsesickness virus serotypes. J Gen Virol 1996;77(Pt 7):1421–3.
    pubmed: 8757982
  43. Martinez-Torrecuadrada JL, Diaz-Laviada M, Roy P, Sanchez C, Vela C, Sanchez-Vizcaino JM. Serologic markers in early stages of African horse sickness virus infection. J Clin Microbiol 1997;35(2):531–5.
    pmc: PMC229621pubmed: 9003637
  44. Regnard GL, Halley-Stott RP, Tanzer FL, Hitzeroth II, Rybicki EP. High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector. Plant Biotechnol J 2010;8(1):38–46.
    pubmed: 19929900
  45. Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T. Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization. J Gen Virol 2007;88(Pt 5):1460–9.
    pubmed: 17412974
  46. Sainsbury F, Thuenemann EC, Lomonossoff GP. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 2009;7(7):682–93.
    pubmed: 19627561
  47. Shen WJ, Forde BG. Efficient transformation of Agrobacterium spp. By high voltage electroporation. Nucleic Acids Res 1989;17(20):8385.
    pmc: PMC334991pubmed: 2682529
  48. Adams A, Hendrikse M, Rybicki EP, Hitzeroth II. Optimal size of DNA encapsidated by plant produced human papillomavirus pseudovirions. Virology 2023;580:88–97.
    pubmed: 36801669
  49. Mbewana S, Meyers AE, Weber B, Mareledwane V, Ferreira ML, Majiwa PAO. Expression of rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen. BMC Biotechnol 2018;18(1):77.
    pmc: PMC6290525pubmed: 30537953
  50. Wade-Evans AM, Mertens PP, Belsham GJ. Sequence of genome segment 9 of bluetongue virus (serotype 1, South Africa) and expression analysis demonstrating that different forms of VP6 are derived from initiation of protein synthesis at two distinct sites. J Gen Virol 1992;73(Pt 11):3023–6.
    pubmed: 1331303
  51. Waal PJd. The characterization of inner core protein VP6 of African horsesickness virus. 2006.
  52. Atkinson R, Burt F, Rybicki EP, Meyers AE. Plant-produced Crimean-Congo haemorrhagic fever virus nucleoprotein for use in indirect ELISA. J Virol Methods 2016;236:170–7.
    pubmed: 27474493
  53. Fearon SH, Dennis SJ, Hitzeroth II, Rybicki EP, Meyers AE. Plant expression systems as an economical alternative for the production of iELISA coating antigen AHSV VP7. N Biotechnol 2022;68:48–56.
    pubmed: 35114407
  54. Gunter CJ, Regnard GL, Rybicki EP, Hitzeroth II. Immunogenicity of plant-produced Porcine circovirus-like particles in mice. Plant Biotechnol J 2019;17(9):1751–9.
    pmc: PMC6686138pubmed: 30791210
  55. Stander J, Mbewana S, Meyers AE. Plant-Derived Hum Vaccines: Recent Developments. BioDrugs 2022;36(5):573–89.
    pmc: PMC9275545pubmed: 35821564

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