title: Plant-produced Crimean-Congo haemorrhagic fever virus nucleoprotein for use in indirect ELISA.
abstract: Crimean-Congo haemorrhagic fever (CCHF) is a disease of serious public concern caused by the CCHF virus (CCHFV). Anti-CCHFV IgG in humans can be detected using ELISA with native antigen prepared from cell cultures which have been infected with virus or from brain tissue of suckling mice which have been inoculated with virus. However, the preparation of these reagents requires high biosafety levels and is expensive. A safer, more cost-effective recombinantly-produced NP reagent is desirable. Recently, plants have been shown to be a cost-effective and safe system for expression of recombinant proteins. This work describes cloning of the CCHFV NP gene into three different plant expression systems and comparison of expression in Nicotiana benthamiana. The highest expressing construct was selected. Expressed NP was purified by ammonium sulphate fractionation prior to histidine affinity chromatography. Purified NP was tested in an indirect ELISA to determine if the recombinant antigen was able to detect anti-CCHFV IgG in sera from convalescent patients. Plant-produced NP detected IgG antibodies against CCHFV in 13/13 serum samples from convalescent patients and 0/13 samples collected from volunteers with no history of CCHFV infection. Results were compared with commercially available immunofluorescent assays and 100% concordance was obtained between the two assays. This suggests that a full evaluation of the plant produced NP for application as a safe recombinant is warranted.
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, P Bag X3, Rondebosch 7701, South Africa. Electronic address: Richard.Atkinson@fco.gov.uk.
authors.forename: Richard
authors.initials: R
authors.lastname: Atkinson
authors.affiliation: Department of Medical Microbiology and Virology, National Health Laboratory Service Universitas and Faculty of Health Sciences, University of Free State, P O Box 339, Bloemfontein 9300, South Africa. Electronic address: BurtFJ@ufs.ac.za.
authors.forename: Felicity
authors.initials: F
authors.lastname: Burt
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, P Bag X3, Rondebosch 7701, South Africa; Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa. Electronic address: Ed.rybicki@uct.ac.za.
authors.forename: Edward P
authors.initials: EP
authors.lastname: Rybicki
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, P Bag X3, Rondebosch 7701, South Africa. Electronic address: Ann.meyers@uct.ac.za.
authors.forename: Ann E
authors.initials: AE
authors.lastname: Meyers
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006479:Hemorrhagic Fever, Crimean / Q000175:diagnosis*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D009698:Nucleoproteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D012698:Serologic Tests / Q000379:methods*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D007074:Immunoglobulin G; D009698:Nucleoproteins; D011994:Recombinant Proteins
keywords: Crimean-Congo haemorrhagic fever; Nucleoprotein; Recombinant antigen