{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T15:32:56Z","timestamp":1783524776202,"version":"3.55.0"},"reference-count":55,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2025,8,12]],"date-time":"2025-08-12T00:00:00Z","timestamp":1754956800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2025,8,12]],"date-time":"2025-08-12T00:00:00Z","timestamp":1754956800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100001323","name":"Poliomyelitis Research Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001323","id-type":"DOI","asserted-by":"publisher"}]},{"name":"University of Cape Town"},{"DOI":"10.13039\/501100001322","name":"South African Medical Research Council","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001322","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Virol J"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>Culicoides<\/jats:italic>\n                      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\n                      <jats:italic>Nicotiana benthamiana<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      AHSV-VP6 sequences from all nine serotypes were aligned to obtain a consensus sequence, which was then used to design the\n                      <jats:italic>VP6<\/jats:italic>\n                      gene. The\n                      <jats:italic>VP6<\/jats:italic>\n                      gene was successfully expressed in\n                      <jats:italic>Nicotiana benthamiana<\/jats:italic>\n                      plants via\n                      <jats:italic>Agrobacterium<\/jats:italic>\n                      -mediated infiltration. The VP6 protein was extracted from infiltrated leaves and purified. A purified yield of approximately 7.7\u00a0mg of recombinant VP6\/kg fresh weight leaf material was obtained. The VP6 protein was also expressed in\n                      <jats:italic>E. coli<\/jats:italic>\n                      , yielding a purified product of 9.4\u00a0mg\/L. Preliminary data revealed that AHSV-VP6 antigen expressed in both plants and\n                      <jats:italic>E. coli<\/jats:italic>\n                      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\n                      <jats:italic>E. coli-<\/jats:italic>\n                      produced VP6.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12985-025-02898-1","type":"journal-article","created":{"date-parts":[[2025,8,12]],"date-time":"2025-08-12T11:59:57Z","timestamp":1754999997000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development of an African horse sickness VP6 DIVA diagnostic ELISA"],"prefix":"10.1186","volume":"22","author":[{"given":"Munyaradzi","family":"Tinarwo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susan J.","family":"Dennis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Inga I.","family":"Hitzeroth","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann E.","family":"Meyers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edward P.","family":"Rybicki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandiswa","family":"Mbewana","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,8,12]]},"reference":[{"key":"2898_CR1","unstructured":"OIE. African horse sickness (Infection with African horse sickness virus) 2019 [Available from: https:\/\/www.oie.int\/standard-setting\/terrestrial-manual\/access-online"},{"issue":"4","key":"2898_CR2","doi-asserted-by":"publisher","first-page":"445","DOI":"10.1051\/vetres:2004021","volume":"35","author":"PS Mellor","year":"2004","unstructured":"Mellor PS, Hamblin C. African horse sickness. Vet Res. 2004;35(4):445\u201366.","journal-title":"Vet Res"},{"issue":"2","key":"2898_CR3","doi-asserted-by":"publisher","first-page":"315","DOI":"10.20506\/rst.34.2.2359","volume":"34","author":"S Zientara","year":"2015","unstructured":"Zientara S, Weyer CT, Lecollinet S. African horse sickness. Sci Tech Rev Office Int Des Epizooties (Paris). 2015;34(2):315\u201327.","journal-title":"Sci Tech Rev Office Int Des Epizooties (Paris)"},{"key":"2898_CR4","doi-asserted-by":"crossref","unstructured":"Dennis SJ, Meyers AE, Hitzeroth II, Rybicki EP. African horse sickness: A review of current Understanding and vaccine development. Viruses. 2019;11(9).","DOI":"10.3390\/v11090844"},{"issue":"1","key":"2898_CR5","doi-asserted-by":"publisher","first-page":"197","DOI":"10.20506\/rst.33.1.2272","volume":"33","author":"S Abul Rahman","year":"2014","unstructured":"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\u2013202.","journal-title":"Rev Sci Tech"},{"key":"2898_CR6","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1146\/annurev.pu.17.050196.001335","volume":"17","author":"AM Beck","year":"1996","unstructured":"Beck AM, Meyers NM. Health enhancement and companion animal ownership. Annu Rev Public Health. 1996;17:247\u201357.","journal-title":"Annu Rev Public Health"},{"key":"2898_CR7","doi-asserted-by":"crossref","unstructured":"Suggett RH. Horses and the rural economy in the united Kingdom. Equine Vet J Suppl. 1999(28):31\u20137.","DOI":"10.1111\/j.2042-3306.1999.tb05153.x"},{"key":"2898_CR8","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1146\/annurev-ento-031616-035010","volume":"62","author":"S Carpenter","year":"2017","unstructured":"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\u201358.","journal-title":"Ann Rev Entomol"},{"key":"2898_CR9","doi-asserted-by":"publisher","first-page":"793","DOI":"10.1099\/0022-1317-71-4-793","volume":"71","author":"CW Bremer","year":"1990","unstructured":"Bremer CW, Huismans H, Van Dijk AA. Characterization and cloning of the African horsesickness virus genome. J Gen Virol. 1990;71:793\u20139.","journal-title":"J Gen Virol"},{"issue":"2","key":"2898_CR10","doi-asserted-by":"publisher","first-page":"108","DOI":"10.1016\/j.prevetmed.2012.01.019","volume":"106","author":"CJ de Vos","year":"2012","unstructured":"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\u201322.","journal-title":"Prev Vet Med"},{"issue":"8","key":"2898_CR11","doi-asserted-by":"publisher","first-page":"2208","DOI":"10.3201\/eid2708.210004","volume":"27","author":"N Bunpapong","year":"2021","unstructured":"Bunpapong N, Charoenkul K, Nasamran C, Chamsai E, Udom K, Boonyapisitsopa S, et al. African horse sickness virus serotype 1 on horse farm, thailand, 2020. Emerg Infect Dis. 2021;27(8):2208\u201311.","journal-title":"Emerg Infect Dis"},{"issue":"5","key":"2898_CR12","first-page":"1764","volume":"67","author":"S King","year":"2020","unstructured":"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\u20137.","journal-title":"Transbound Emerg Dis"},{"issue":"5","key":"2898_CR13","doi-asserted-by":"publisher","first-page":"e0252117","DOI":"10.1371\/journal.pone.0252117","volume":"16","author":"JD Grewar","year":"2021","unstructured":"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.","journal-title":"PLoS ONE"},{"issue":"12","key":"2898_CR14","doi-asserted-by":"publisher","first-page":"2087","DOI":"10.3201\/eid2212.160718","volume":"22","author":"CT Weyer","year":"2016","unstructured":"Weyer CT, Grewar JD, Burger P, Rossouw E, Lourens C, Joone C, et al. African horse sickness caused by genome reassortment and reversion to virulence of live, attenuated vaccine viruses, South africa, 2004\u20132014. Emerg Infect Dis. 2016;22(12):2087\u201396.","journal-title":"Emerg Infect Dis"},{"issue":"Pt 9","key":"2898_CR15","doi-asserted-by":"publisher","first-page":"2053","DOI":"10.1099\/0022-1317-77-9-2053","volume":"77","author":"P Roy","year":"1996","unstructured":"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\u20137.","journal-title":"J Gen Virol"},{"issue":"39","key":"2898_CR16","doi-asserted-by":"publisher","first-page":"4932","DOI":"10.1016\/j.vaccine.2014.07.031","volume":"32","author":"Y Kanai","year":"2014","unstructured":"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\u20137.","journal-title":"Vaccine"},{"issue":"46","key":"2898_CR17","doi-asserted-by":"publisher","first-page":"7003","DOI":"10.1016\/j.vaccine.2018.09.065","volume":"36","author":"M Aksular","year":"2018","unstructured":"Aksular M, Calvo-Pinilla E, Marin-Lopez A, Ortego J, Chambers AC, King LA, et al. 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\u201310.","journal-title":"Vaccine"},{"issue":"4","key":"2898_CR18","doi-asserted-by":"publisher","first-page":"e0301340","DOI":"10.1371\/journal.pone.0301340","volume":"19","author":"MM O\u2019Kennedy","year":"2024","unstructured":"O\u2019Kennedy MM, Roth R, Ebersohn K, du Plessis LH, Mamputha S, Rutkowska DA, et al. Immunogenic profile of a plant-produced nonavalent African horse sickness viral protein 2 (VP2) vaccine in IFNAR-\/- mice. PLoS ONE. 2024;19(4):e0301340.","journal-title":"PLoS ONE"},{"issue":"2","key":"2898_CR19","first-page":"0030","volume":"66","author":"M Romito","year":"1999","unstructured":"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\u20132465. (Print)).","journal-title":"Onderstepoort J Veterinaty Rep"},{"issue":"1","key":"2898_CR20","doi-asserted-by":"publisher","first-page":"219","DOI":"10.1006\/viro.1996.0304","volume":"220","author":"MA Stone-Marschat","year":"1996","unstructured":"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\u201322.","journal-title":"Virology"},{"issue":"33","key":"2898_CR21","doi-asserted-by":"publisher","first-page":"4434","DOI":"10.1016\/j.vaccine.2009.05.044","volume":"27","author":"AJ Guthrie","year":"2009","unstructured":"Guthrie AJ, Quan M, Lourens CW, Audonnet JC, Minke JM, Yao J, et al. 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\u20138.","journal-title":"Vaccine"},{"key":"2898_CR22","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/j.virusres.2013.12.002","volume":"180","author":"E Calvo-Pinilla","year":"2014","unstructured":"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\u201330.","journal-title":"Virus Res"},{"issue":"17","key":"2898_CR23","doi-asserted-by":"publisher","first-page":"8764","DOI":"10.1128\/JVI.01052-15","volume":"89","author":"SG van de Water","year":"2015","unstructured":"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\u201372.","journal-title":"J Virol"},{"issue":"33","key":"2898_CR24","doi-asserted-by":"publisher","first-page":"4262","DOI":"10.1016\/j.vaccine.2017.06.023","volume":"35","author":"V Lulla","year":"2017","unstructured":"Lulla V, Losada A, Lecollinet S, Kerviel A, Lilin T, Sailleau C, et al. Protective efficacy of multivalent replication-abortive vaccine strains in horses against African horse sickness virus challenge. Vaccine. 2017;35(33):4262\u20139.","journal-title":"Vaccine"},{"issue":"15","key":"2898_CR25","doi-asserted-by":"publisher","first-page":"1925","DOI":"10.1016\/j.vaccine.2018.03.003","volume":"36","author":"PA van Rijn","year":"2018","unstructured":"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\u201333.","journal-title":"Vaccine"},{"key":"2898_CR26","doi-asserted-by":"publisher","first-page":"179","DOI":"10.1016\/0166-0934(93)90102-W","volume":"45","author":"AM Wade-Evans","year":"1993","unstructured":"Wade-Evans AM, Woolhouse T, O\u2019Hara 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\u201388.","journal-title":"J Virol Methods"},{"issue":"4","key":"2898_CR27","first-page":"0030","volume":"65","author":"M du Plessis","year":"1998","unstructured":"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\u20132465. (Print)).","journal-title":"Onderstepoort J Veterinary Res"},{"key":"2898_CR28","doi-asserted-by":"publisher","first-page":"184","DOI":"10.1016\/j.virusres.2015.12.006","volume":"213","author":"S Maree","year":"2016","unstructured":"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\u201394.","journal-title":"Virus Res"},{"issue":"2","key":"2898_CR29","doi-asserted-by":"publisher","first-page":"442","DOI":"10.1111\/pbi.12783","volume":"16","author":"SJ Dennis","year":"2018","unstructured":"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\u201350.","journal-title":"Plant Biotechnol J"},{"issue":"1","key":"2898_CR30","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1186\/s13567-018-0600-4","volume":"49","author":"SJ Dennis","year":"2018","unstructured":"Dennis SJ, O\u2019Kennedy MM, Rutkowska D, Tsekoa T, Lourens CW, Hitzeroth II, et al. Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses. Vet Res. 2018;49(1):105.","journal-title":"Vet Res"},{"issue":"1","key":"2898_CR31","doi-asserted-by":"publisher","first-page":"432","DOI":"10.1186\/s12917-019-2184-2","volume":"15","author":"DA Rutkowska","year":"2019","unstructured":"Rutkowska DA, Mokoena NB, Tsekoa TL, Dibakwane VS, O\u2019Kennedy MM. Plant-produced chimeric virus-like particles - a new generation vaccine against African horse sickness. BMC Vet Res. 2019;15(1):432.","journal-title":"BMC Vet Res"},{"key":"2898_CR32","unstructured":"Dennis SJ. Development of plant-produced African horse sickness virus vaccines. University of Cape Town; 2019."},{"issue":"Pt 4","key":"2898_CR33","doi-asserted-by":"publisher","first-page":"925","DOI":"10.1099\/0022-1317-73-4-925","volume":"73","author":"T Chuma","year":"1992","unstructured":"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\u201331.","journal-title":"J Gen Virol"},{"issue":"3","key":"2898_CR34","doi-asserted-by":"publisher","first-page":"873","DOI":"10.20506\/rst.12.3.733","volume":"12","author":"LA Oladosu","year":"1993","unstructured":"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\u20137.","journal-title":"Nigeria Rev Sci Tech"},{"key":"2898_CR35","unstructured":"Tessler J. Detection of African horsesickness viral antigens in tissues by Immunofluorescence. Can J Comp Med. 1972;36(2)(0008-4050 (Print))."},{"key":"2898_CR36","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1016\/j.jviromet.2019.01.016","volume":"266","author":"A van Schalkwyk","year":"2019","unstructured":"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\u201394.","journal-title":"J Virol Methods"},{"issue":"12","key":"2898_CR37","doi-asserted-by":"publisher","first-page":"2446","DOI":"10.3201\/eid2812.220594","volume":"28","author":"Y Wang","year":"2022","unstructured":"Wang Y, Ong J, Ng OW, Songkasupa T, Koh EY, Wong JPS, et al. 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\u201354.","journal-title":"Emerg Infect Dis"},{"key":"2898_CR38","doi-asserted-by":"crossref","unstructured":"Laegreid WW. Diagnosis of African horse sickness. Comp Immunol Microbiol Infect Dis. 1994;17(3):297\u2013303.","DOI":"10.1016\/0147-9571(94)90049-3"},{"issue":"1","key":"2898_CR39","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1016\/j.jviromet.2004.12.002","volume":"125","author":"S Maree","year":"2005","unstructured":"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\u201365.","journal-title":"J Virol Methods"},{"key":"2898_CR40","doi-asserted-by":"publisher","first-page":"303","DOI":"10.1017\/S0950268800059483","volume":"104","author":"C Hamblin","year":"1990","unstructured":"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\u201312.","journal-title":"Epidemiol Infect"},{"key":"2898_CR41","volume-title":"Validation of ELISA for the detection of African horse sickness virus antigens and antibodies","year":"1998","unstructured":"Rubio C, Cubillo MA, Hooghuis H, Sanchez-Vizcaino JM, Diaz-Laviada M, Plateau E, et al. editors. Validation of ELISA for the detection of African horse sickness virus antigens and antibodies. Vienna: Springer Vienna; 1998."},{"issue":"Pt 7","key":"2898_CR42","doi-asserted-by":"publisher","first-page":"1421","DOI":"10.1099\/0022-1317-77-7-1421","volume":"77","author":"PJ Turnbull","year":"1996","unstructured":"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\u20133.","journal-title":"J Gen Virol"},{"issue":"2","key":"2898_CR43","doi-asserted-by":"publisher","first-page":"531","DOI":"10.1128\/jcm.35.2.531-535.1997","volume":"35","author":"JL Martinez-Torrecuadrada","year":"1997","unstructured":"Martinez-Torrecuadrada JL, Diaz-Laviada M, Roy P, Sanchez C, Vela C, Sanchez-Vizcaino JM, et al. Serologic markers in early stages of African horse sickness virus infection. J Clin Microbiol. 1997;35(2):531\u20135.","journal-title":"J Clin Microbiol"},{"issue":"1","key":"2898_CR44","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1111\/j.1467-7652.2009.00462.x","volume":"8","author":"GL Regnard","year":"2010","unstructured":"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\u201346.","journal-title":"Plant Biotechnol J"},{"issue":"Pt 5","key":"2898_CR45","doi-asserted-by":"publisher","first-page":"1460","DOI":"10.1099\/vir.0.82718-0","volume":"88","author":"J Maclean","year":"2007","unstructured":"Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T, et al. 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\u20139.","journal-title":"J Gen Virol"},{"issue":"7","key":"2898_CR46","doi-asserted-by":"publisher","first-page":"682","DOI":"10.1111\/j.1467-7652.2009.00434.x","volume":"7","author":"F Sainsbury","year":"2009","unstructured":"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\u201393.","journal-title":"Plant Biotechnol J"},{"issue":"20","key":"2898_CR47","doi-asserted-by":"publisher","first-page":"8385","DOI":"10.1093\/nar\/17.20.8385","volume":"17","author":"WJ Shen","year":"1989","unstructured":"Shen WJ, Forde BG. Efficient transformation of Agrobacterium spp. By high voltage electroporation. Nucleic Acids Res. 1989;17(20):8385.","journal-title":"Nucleic Acids Res"},{"key":"2898_CR48","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1016\/j.virol.2023.02.003","volume":"580","author":"A Adams","year":"2023","unstructured":"Adams A, Hendrikse M, Rybicki EP, Hitzeroth II. Optimal size of DNA encapsidated by plant produced human papillomavirus pseudovirions. Virology. 2023;580:88\u201397.","journal-title":"Virology"},{"issue":"1","key":"2898_CR49","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1186\/s12896-018-0489-z","volume":"18","author":"S Mbewana","year":"2018","unstructured":"Mbewana S, Meyers AE, Weber B, Mareledwane V, Ferreira ML, Majiwa PAO, et al. Expression of rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen. BMC Biotechnol. 2018;18(1):77.","journal-title":"BMC Biotechnol"},{"issue":"Pt 11","key":"2898_CR50","doi-asserted-by":"publisher","first-page":"3023","DOI":"10.1099\/0022-1317-73-11-3023","volume":"73","author":"AM Wade-Evans","year":"1992","unstructured":"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\u20136.","journal-title":"J Gen Virol"},{"key":"2898_CR51","unstructured":"Waal PJd. The characterization of inner core protein VP6 of African horsesickness virus. University of Pretoria; 2006."},{"key":"2898_CR52","doi-asserted-by":"publisher","first-page":"170","DOI":"10.1016\/j.jviromet.2016.07.025","volume":"236","author":"R Atkinson","year":"2016","unstructured":"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\u20137.","journal-title":"J Virol Methods"},{"key":"2898_CR53","doi-asserted-by":"publisher","first-page":"48","DOI":"10.1016\/j.nbt.2022.01.009","volume":"68","author":"SH Fearon","year":"2022","unstructured":"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\u201356.","journal-title":"N Biotechnol"},{"issue":"9","key":"2898_CR54","doi-asserted-by":"publisher","first-page":"1751","DOI":"10.1111\/pbi.13097","volume":"17","author":"CJ Gunter","year":"2019","unstructured":"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\u20139.","journal-title":"Plant Biotechnol J"},{"issue":"5","key":"2898_CR55","first-page":"573","volume":"36","author":"J Stander","year":"2022","unstructured":"Stander J, Mbewana S, Meyers AE. Plant-Derived Hum Vaccines: Recent Developments BioDrugs. 2022;36(5):573\u201389.","journal-title":"Plant-Derived Hum Vaccines: Recent Developments BioDrugs"}],"container-title":["Virology Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12985-025-02898-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12985-025-02898-1","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12985-025-02898-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T11:13:43Z","timestamp":1781608423000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1186\/s12985-025-02898-1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,12]]},"references-count":55,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["2898"],"URL":"https:\/\/doi.org\/10.1186\/s12985-025-02898-1","relation":{},"ISSN":["1743-422X"],"issn-type":[{"value":"1743-422X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,12]]},"assertion":[{"value":"7 April 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"26 July 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"12 August 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"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.\n                      FHS AEC REF NO: 020_025.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"276"}}