{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T22:55:00Z","timestamp":1789167300275,"version":"build-2803163510"},"reference-count":57,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2022,9,11]],"date-time":"2022-09-11T00:00:00Z","timestamp":1662854400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Prof M. Venter\u2019s University of Pretoria Development Fund","award":["115574"],"award-info":[{"award-number":["115574"]}]},{"name":"Prof M. Venter\u2019s University of Pretoria Development Fund","award":["FKZ1368-1438TO08"],"award-info":[{"award-number":["FKZ1368-1438TO08"]}]},{"name":"EU\/NRF LEAP AGRI LEARN project","award":["115574"],"award-info":[{"award-number":["115574"]}]},{"name":"EU\/NRF LEAP AGRI LEARN project","award":["FKZ1368-1438TO08"],"award-info":[{"award-number":["FKZ1368-1438TO08"]}]},{"name":"G7 Global Health Fund program","award":["115574"],"award-info":[{"award-number":["115574"]}]},{"name":"G7 Global Health Fund program","award":["FKZ1368-1438TO08"],"award-info":[{"award-number":["FKZ1368-1438TO08"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Although Old World alphaviruses, Middelburg- (MIDV) and Sindbis virus (SINV), have previously been detected in horses and wildlife with neurologic disease in South Africa, the pathogenesis and clinical presentation of MIDV and SINV infections in animals are not well documented. Clinical samples from horses across South Africa with acute or fatal neurologic and febrile infections submitted between 2014\u20132018 were investigated. In total, 69\/1084 (6.36%) and 11\/1084 (1.01%) horses tested positive for MIDV and SINV, respectively, by real-time reverse transcription (RT) PCR. Main signs\/outcomes for MIDV (n = 69): 73.91% neurological, 75.36% fever, 28.99% icterus and anorexia, respectively, 8.70% fatalities; SINV (n = 11): 54.54% neurological, 72.73% fever, 36.36% anorexia and 18.18% fatalities. MIDV cases peaked in the late summer\/autumn across most South African provinces while SINV cases did not show a clear seasonality and were detected in fewer South African provinces. MIDV could still be detected in blood samples via RT-PCR for up to 71,417 and 21 days after onset of signs in 4 horses respectively, suggesting prolonged replication relative to SINV which could only be detected in the initial sample. Phylogenetic analyses based on partial sequences of the nsP4 (MIDV n = 59 and SINV n = 7) and E1 (MIDV n = 45) genes, as well as full genome sequences (MIDV n = 6), clustered the MIDV and SINV strains from the present study with previously detected strains. MIDV infection appears to be more prevalent in horses than SINV infection based on RT-PCR results, however, prevalence estimates might be different when also considering serological surveillance data.<\/jats:p>","DOI":"10.3390\/v14092013","type":"journal-article","created":{"date-parts":[[2022,9,14]],"date-time":"2022-09-14T00:15:02Z","timestamp":1663114502000},"page":"2013","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014\u20132018)"],"prefix":"10.3390","volume":"14","author":[{"given":"Isabel","family":"Fourie","sequence":"first","affiliation":[{"name":"Department of Medical Virology, University of Pretoria, Pretoria 0031, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4647-8934","authenticated-orcid":false,"given":"Jumari","family":"Snyman","sequence":"additional","affiliation":[{"name":"Department of Medical Virology, University of Pretoria, Pretoria 0031, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"June","family":"Williams","sequence":"additional","affiliation":[{"name":"Department of Paraclinical Sciences, University of Pretoria, Pretoria 0110, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4672-5915","authenticated-orcid":false,"given":"Arshad","family":"Ismail","sequence":"additional","affiliation":[{"name":"Sequencing Core Facility, National Institute of Communicable Diseases (NICD), Johannesburg 2192, South Africa"},{"name":"Department of Biochemistry and Microbiology, University of Venda, Thohoyandou 0950, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2862-7983","authenticated-orcid":false,"given":"Petrus","family":"Jansen van Vuren","sequence":"additional","affiliation":[{"name":"Australian Centre for Disease Preparedness, CSIRO-Health and Biosecurity, Geelong 3220, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2696-824X","authenticated-orcid":false,"given":"Marietjie","family":"Venter","sequence":"additional","affiliation":[{"name":"Department of Medical Virology, University of Pretoria, Pretoria 0031, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"732","DOI":"10.1056\/NEJMoa1212628","article-title":"Eastern equine encephalitis in Latin America","volume":"369","author":"Carrera","year":"2013","journal-title":"N. Engl. J. Med."},{"key":"ref_2","first-page":"163","article-title":"Mayaro: An emerging viral threat?","volume":"7","author":"Monsalve","year":"2018","journal-title":"Emerg. Microbes. Infect."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"189","DOI":"10.7589\/0090-3558-29.2.189","article-title":"Viremia in three orders of brids (Anseriforms, Galliformes and Passeriformes) inoculated with Ockelbo virus","volume":"29","author":"Lundstrom","year":"1993","journal-title":"J. Wildl. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1002\/rmv.1876","article-title":"Sindbis virus as a human pathogen-epidemiology, clinical picture and pathogenesis","volume":"26","author":"Adouchief","year":"2016","journal-title":"Rev. Med. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"844","DOI":"10.4269\/ajtmh.1955.4.844","article-title":"Sindbis Virus: A Newly Recognized Arthropod-Transmitted Virus1","volume":"4","author":"Taylor","year":"1955","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_6","first-page":"295","article-title":"Epidemics of West Nile and Sindbis viruses in South Africa with Culex (Culex) univittatus Theobald as vector","volume":"72","author":"McIntosh","year":"1976","journal-title":"South Afr. J. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1017\/S0950268813000964","article-title":"Human cases of Sindbis fever in South Africa, 2006-2010","volume":"142","author":"Storm","year":"2014","journal-title":"Epidemiol. Infect."},{"key":"ref_8","unstructured":"Centre for Emerging ZaPD, NICD-NHLS (2017). Cluster of Sindbis virus infections in Gauteng Province: An update. Division of Public Health Surveillance and Response, NICD Division of Public Health Surveillance and Response."},{"key":"ref_9","first-page":"145","article-title":"Middelburg virus; a hitherto unknown agent isolated from Aedes mosquitoes during an epizootic in sheep in the eastern Cape Province","volume":"22","author":"Kokernot","year":"1957","journal-title":"S. Afr. J. Med. Sci."},{"key":"ref_10","unstructured":"McIntosh, B.M. (1980). The Epidemiology of Arthropod-Borne Viruses in Southern Africa Pretoria (SA). [Doctoral Dissertation, University of Pretoria]."},{"key":"ref_11","unstructured":"Human, S., Steyl, J., Williams, J., Last, R., van Niekerk, S., and Venter, M. (2010, January 18\u201320). Sindbis and Middelburg Viruses as a Cause of Disease in Animals in South Africa: The Molecular Epidemiology. Proceedings of the 9th Annual Congress of the Southern African Society for Veterinary Epidemiology and Preventive Medicine, Farm Inn, Pretoria, South Africa."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3078","DOI":"10.1099\/vir.0.83076-0","article-title":"Complete nucleotide sequence of Middelburg virus, isolated from the spleen of a horse with severe clinical disease in Zimbabwe","volume":"88","author":"Attoui","year":"2007","journal-title":"J. Gen. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2225","DOI":"10.3201\/eid2112.150132","article-title":"Sindbis and Middelburg Old World Alphaviruses Associated with Neurologic Disease in Horses, South Africa","volume":"21","author":"Human","year":"2015","journal-title":"Emerg. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1182","DOI":"10.3201\/eid2606.191179","article-title":"Zoonotic Alphaviruses in Fatal and Neurologic Infections in Wildlife and Nonequine Domestic Animals, South Africa","volume":"26","author":"Steyn","year":"2020","journal-title":"Emerg. Infect. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1016\/S0166-0934(01)00306-8","article-title":"A generic nested-RT-PCR followed by sequencing for detection and identification of members of the alphavirus genus","volume":"95","author":"Rosario","year":"2001","journal-title":"J. Virol. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/j.jviromet.2008.12.014","article-title":"A highly sensitive method for the detection and genotyping of West Nile virus by real-time PCR","volume":"157","author":"Zaayman","year":"2009","journal-title":"J. Virol. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2883","DOI":"10.1007\/s00705-014-2131-2","article-title":"Genomic and phylogenetic characterization of Shuni virus","volume":"159","author":"Harders","year":"2014","journal-title":"Arch Virol."},{"key":"ref_18","first-page":"207","article-title":"Phylogeny of Sindbis virus isolates from South Africa","volume":"28","author":"Storm","year":"2013","journal-title":"South. Afr. J. Infect. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Jansen van Vuren, P., Wiley, M., Palacios, G., Storm, N., McCulloch, S., Markotter, W., Birkhead, M., Kemp, A., and Paweska, J.T. (2016). Isolation of a Novel Fusogenic Orthoreovirus from Eucampsipoda africana Bat Flies in South Africa. Viruses, 8.","DOI":"10.3390\/v8030065"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2729","DOI":"10.1128\/JVI.05591-11","article-title":"Genome-scale phylogeny of the alphavirus genus suggests a marine origin","volume":"86","author":"Forrester","year":"2012","journal-title":"J. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"14622","DOI":"10.1073\/pnas.1204787109","article-title":"Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication","volume":"109","author":"Nasar","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1160","DOI":"10.1093\/bib\/bbx108","article-title":"MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization","volume":"20","author":"Katoh","year":"2017","journal-title":"Brief. Bioinform."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1038\/nmeth.2109","article-title":"jModelTest 2: More models, new heuristics and parallel computing","volume":"9","author":"Darriba","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Drummond, A.J., and Rambaut, A. (2007). BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol., 7.","DOI":"10.1186\/1471-2148-7-214"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Miller, M.A., Pfeiffer, W., and Schwartz, T. (2010, January 14). Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Proceedings of the 2010 Gateway Computing Environments Workshop (GCE), New Orleans, LA, USA.","DOI":"10.1109\/GCE.2010.5676129"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"St\u00f6ver, B.C., and M\u00fcller, K.F. (2010). TreeGraph 2: Combining and visualizing evidence from different phylogenetic analyses. BMC Bioinform., 11.","DOI":"10.1186\/1471-2105-11-7"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1093\/molbev\/msw054","article-title":"MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets","volume":"33","author":"Kumar","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_28","unstructured":"Dean, A.G., Arner, T.G., Sunki, G.G., Friedman, R., Lantinga, M., Sangam, S., Zubieta, J.C., Sullivan, K.M., Brendel, K.A., and Gao, Z. (2019, November 20). Epi Info\u2122, a Database and Statistics Program for Public Health Professionals, Available online: https:\/\/www.cdc.gov\/epiinfo."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"vev003","DOI":"10.1093\/ve\/vev003","article-title":"RDP4: Detection and analysis of recombination patterns in virus genomes","volume":"1","author":"Martin","year":"2015","journal-title":"Virus Evol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1093\/bioinformatics\/16.6.562","article-title":"RDP: Detection of recombination amongst aligned sequences","volume":"16","author":"Martin","year":"2000","journal-title":"Bioinformatics"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1006\/viro.1999.0056","article-title":"Possible emergence of new geminiviruses by frequent recombination","volume":"265","author":"Padidam","year":"1999","journal-title":"Virology"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"708","DOI":"10.1093\/oxfordjournals.molbev.a004129","article-title":"Evaluation of methods for detecting recombination from DNA sequences: Empirical data","volume":"19","author":"Posada","year":"2002","journal-title":"Mol. Biol. Evol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1093\/bioinformatics\/16.7.573","article-title":"Sister-scanning: A Monte Carlo procedure for assessing signals in recombinant sequences","volume":"16","author":"Gibbs","year":"2000","journal-title":"Bioinformatics"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1089\/aid.2005.21.98","article-title":"A modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints","volume":"21","author":"Martin","year":"2005","journal-title":"AIDS Res. Hum. Retrovir."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1007\/BF00182389","article-title":"Analyzing the mosaic structure of genes","volume":"34","author":"Smith","year":"1992","journal-title":"J. Mol. Evol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1093\/molbev\/msx263","article-title":"Improved Algorithmic Complexity for the 3SEQ Recombination Detection Algorithm","volume":"35","author":"Lam","year":"2018","journal-title":"Mol. Biol. Evol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e01078-14","DOI":"10.1128\/genomeA.01078-14","article-title":"Complete genome sequences of two middelburg viruses isolated from arthropods in the central african republic","volume":"2","author":"Tricou","year":"2014","journal-title":"Genome Announc."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.coviro.2017.11.004","article-title":"Assessing the zoonotic potential of arboviruses of African origin","volume":"28","author":"Venter","year":"2018","journal-title":"Curr. Opin. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Fourie, I., Williams, J., Ismail, A., Jansen van Vuren, P., Stoltz, A., and Venter, M. (2022). Detection and genome characterization of Middelburg virus strains isolated from CSF and whole blood samples of humans with neurological manifestations in South Africa. PLoS Negl. Trop. Dis., 16.","DOI":"10.1371\/journal.pntd.0010020"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1016\/j.jviromet.2011.10.021","article-title":"Development and evaluation of a real-time RT-PCR assay for Sindbis virus detection","volume":"179","author":"Sane","year":"2012","journal-title":"J. Virol. Methods"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2243","DOI":"10.3201\/eid2512.181810","article-title":"Epidemiologic, Entomologic, and Virologic Factors of the 2014-15 Ross River Virus Outbreak, Queensland, Australia","volume":"25","author":"Jansen","year":"2019","journal-title":"Emerg. Infect. Dis."},{"key":"ref_42","unstructured":"Maramorosch, K., Murphy, F.A., and Shatkin, A.J. (1989). Eastern Equine Encephalomyelitis Virus: Epidemiology and Evolution of Mosquito Transmission, Academic Press. Advances in Virus Research."},{"key":"ref_43","first-page":"218","article-title":"Sindbis and West Nile virus infections in the Witwatersrand-Pretoria region","volume":"70","author":"Jupp","year":"1986","journal-title":"S. Afr. Med. J."},{"key":"ref_44","first-page":"393","article-title":"Isolations of Middelburg virus from Aedes (Ochlerotatus) juppi McIntosh (Diptera: Culicidae) suggestive of a reservoir vector","volume":"50","author":"Jupp","year":"1987","journal-title":"J. Entomol. Soc. South. Afr."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1214","DOI":"10.2307\/2260340","article-title":"Vegetation Map of Africa. The Vegetation of Africa: A Descriptive Memoir to Accompany the Unesco\/AETFAT\/UNSO Vegetation map of Africa","volume":"75","author":"Hopkins","year":"1987","journal-title":"J. Ecol."},{"key":"ref_46","unstructured":"(2017). South African Weather Service Annual Report 2016\/2017 [Press Release], South African Weather Service."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Lim, E.X.Y., Lee, W.S., Madzokere, E.T., and Herrero, L.J. (2018). Mosquitoes as Suitable Vectors for Alphaviruses. Viruses, 10.","DOI":"10.3390\/v10020084"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"837","DOI":"10.2217\/fmb.09.59","article-title":"A structural and functional perspective of alphavirus replication and assembly","volume":"4","author":"Jose","year":"2009","journal-title":"Future Microbiol."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Tsetsarkin, K.A., Vanlandingham, D.L., McGee, C.E., and Higgs, S. (2007). A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog., 3.","DOI":"10.1371\/journal.ppat.0030201"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1186\/s13071-017-2559-9","article-title":"Mosquito-borne arboviruses of African origin: Review of key viruses and vectors","volume":"11","author":"Braack","year":"2018","journal-title":"Parasites Vectors"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"9397","DOI":"10.1128\/JVI.79.15.9397-9409.2005","article-title":"Inhibition of transcription and translation in Sindbis virus-infected cells","volume":"79","author":"Gorchakov","year":"2005","journal-title":"J. Virol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/j.virol.2016.06.005","article-title":"Host translation shutoff mediated by non-structural protein 2 is a critical factor in the antiviral state resistance of Venezuelan equine encephalitis virus","volume":"496","author":"Bhalla","year":"2016","journal-title":"Virology"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"386","DOI":"10.4269\/ajtmh.1990.42.386","article-title":"An outbreak of human Semliki Forest virus infections in Central African Republic","volume":"42","author":"Mathiot","year":"1990","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1127","DOI":"10.1126\/science.424742","article-title":"Semliki forest virus: Cause of a fatal case of human encephalitis","volume":"203","author":"Willems","year":"1979","journal-title":"Science"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1128\/jvi.50.1.66-72.1984","article-title":"Pathogenesis of in utero infections with abortogenic and non-abortogenic alphaviruses in mice","volume":"50","author":"Milner","year":"1984","journal-title":"J. Virol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1128\/CMR.00045-12","article-title":"West Nile Virus: Biology, transmission, and human infection","volume":"25","author":"Colpitts","year":"2012","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1111\/jvec.12378","article-title":"Mosquito community composition and abundance at contrasting sites in northern South Africa, 2014\u20132017","volume":"45","author":"Johnson","year":"2020","journal-title":"J. Vector Ecol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/9\/2013\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:29:32Z","timestamp":1760142572000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/9\/2013"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,11]]},"references-count":57,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2022,9]]}},"alternative-id":["v14092013"],"URL":"https:\/\/doi.org\/10.3390\/v14092013","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,11]]}}}