title: Epitope mapping of bovine viral diarrhea virus nonstructural protein 3.
abstract: Six consecutive overlapped coding regions (F1-F6) of whole NS3 molecule of bovine viral diarrhea virus (BVDV) were cloned into pMAL-c2X plasmid vector and expressed in Escherichia coli cells (BL21 strain). The recombinant proteins were then purified by amylose resin to determine the most immunogenic domain(s) of the NS3 molecule. Evaluation of the recombinant proteins was carried out by indirect ELISAs using several bovine sera (previously characterized by virus neutralization test, a commercial ELISA kit, and a newly developed NS3-ELISA) and 6 monoclonal antibodies. The experiments showed that the most immunogenic domain of the NS3 protein was the fourth designed fragment (F4), a 122 amino-acid (AA) region of about 13.5 kDa (nucleotide 1003-1368; residue 335-456). Purified recombinant F4 was also evaluated as single ELISA antigen (F4-ELISA) for the detection of anti-BVDV antibodies in sera of infected cattle. Although this small recombinant fragment of NS3 protein was almost completely soluble and expressed more efficient respect to whole NS3 molecule, it did not show enough sensitivity and specificity to be a proper substitute for NS3 as ELISA antigen to detect specific antibodies against BVDV. However, statistical analyses showed a medium correlation between the results of the developed F4-ELISA and virus neutralization test (kappa coefficient=0.63, P<0.001), with the relative sensitivity and specificity of 78.05% and 84.91%, respectively, suggesting the potential use of this fragment as an ELISA antigen along with other antigens or monoclonal antibody(s) in a competitive ELISA.
authors.affiliation: Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran.
authors.forename: Pezhman
authors.initials: P
authors.lastname: Mahmoodi
authors.affiliation: Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran. Electronic address: masoudrs@scu.ac.ir.
authors.forename: Masoud Reza Seyfi Abad
authors.initials: MR
authors.lastname: Shapouri
authors.affiliation: Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran.
authors.forename: Masoud
authors.initials: M
authors.lastname: Ghorbanpour
authors.affiliation: Department of Pharmacognosy, Faculty of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Ekhtelat
authors.affiliation: Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran.
authors.forename: Mohammad Rahim Haji
authors.initials: MR
authors.lastname: Hajikolaei
authors.affiliation: Razi Vaccine and Serum Research Institute, Karaj, Iran.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Lotfi
authors.affiliation: Department of Food Hygiene, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran.
authors.forename: Mahdi Pourmahdi
authors.initials: MP
authors.lastname: Boroujeni
authors.affiliation: Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Daghari
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000378:metabolism*; D018604:Epitope Mapping / Q000662:veterinary*; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D010447:Peptide Hydrolases; D020365:RNA Helicases; D017361:Viral Nonstructural Proteins; C086730:p80 protein, bovine viral diarrhea virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
keywords: Bovine viral diarrhea; Diagnosis; Epitope; NS3; Serology