title: Production and characterization of a Brazilian candidate antigen for Hepatitis E Virus genotype 3 diagnosis.
abstract: Hepatitis E, caused by hepatitis E virus (HEV), is a viral infectious pathology of great importance in the public health. Hepatitis E outbreaks were registered in developing countries with poor or no sanitation, where drinking water was contaminated with fecal material, but also in many industrialized countries probably due to consumption of HEV-positive swine meat. In this study, we present the development and characterization of a recombinant antigen from ORF2 HEV genotype 3. Viral RNA was extracted from swine feces infected with the native virus. A total of 267 residues from the C-terminal ORF2((394-661)) coding sequence were cloned into the pET20a vector and expressed in Escherichia coli ER2566. Recombinant protein was purified by liquid chromatography and the fragment obtained a 98% homology against other human or swine HEV genotype 3 ORF2 sequences. Wistar rats were inoculated with ORF2p, developing antibodies able to recognize both the homologous antigen and the native HEV genotype 3 ORF2 present in infected stool. In parallel, HEV-negative swine were experimentally challenged with HEV genotype 3. ORF2 was detected by PCR 14 days post-inoculation in three-fourth piglets' feces and one week later by dot blot. In conclusion, this study proved the immunogenic and antigenic properties of the recombinant protein ORF2p.
authors.affiliation: Laboratory of Microbiology and Advanced Immunology, Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo, Passo Fundo, RS, 99052-900, Brazil.
authors.forename: Denise
authors.initials: D
authors.lastname: de Almeida Ramos
authors.affiliation: Laboratory of Microbiology and Advanced Immunology, Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo, Passo Fundo, RS, 99052-900, Brazil.
authors.forename: Michela
authors.initials: M
authors.lastname: Miani
authors.affiliation: Laboratory of Microbiology and Advanced Immunology, Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo, Passo Fundo, RS, 99052-900, Brazil.
authors.forename: Rafael
authors.initials: R
authors.lastname: Pandolfi
authors.affiliation: Laboratory of Microbiology and Advanced Immunology, Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo, Passo Fundo, RS, 99052-900, Brazil.
authors.forename: Luis
authors.initials: L
authors.lastname: Tondo
authors.affiliation: Laboratory of Microbiology and Advanced Immunology, Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo, Passo Fundo, RS, 99052-900, Brazil.
authors.forename: Maikel L
authors.initials: ML
authors.lastname: Colli
authors.affiliation: Molecular Microbiology Laboratory, Institute of Health Sciences, Feevale University, Novo Hamburgo, RS, 93525-075, Brazil.
authors.forename: Fernando
authors.initials: F
authors.lastname: Rosado Spilki
authors.affiliation: Laboratory of Technological Development in Virology, Oswaldo Cruz Institute Foundation, Rio de Janeiro, RJ, 21045-900, Brazil.
authors.forename: Noemi
authors.initials: N
authors.lastname: Rovaris Gardinali
authors.affiliation: Laboratory of Technological Development in Virology, Oswaldo Cruz Institute Foundation, Rio de Janeiro, RJ, 21045-900, Brazil.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Alves Pinto
authors.affiliation: Laboratory of Microbiology and Advanced Immunology, Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo, Passo Fundo, RS, 99052-900, Brazil.
authors.forename: Luiz C
authors.initials: LC
authors.lastname: Kreutz
authors.affiliation: Laboratory of Microbiology and Advanced Immunology, Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo, Passo Fundo, RS, 99052-900, Brazil rfran@upf.br.
authors.forename: Rafael
authors.initials: R
authors.lastname: Frandoloso
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000276:immunology*; D001483:Base Sequence; D001938:Brazil; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005243:Feces / Q000821:virology*; D016751:Hepatitis E / Q000175:diagnosis*; D016752:Hepatitis E virus / Q000145:classification / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA; D013552:Swine; D013553:Swine Diseases
chemical_list: D000956:Antigens, Viral; D012367:RNA, Viral
keywords: ORF2 protein; diagnosis; hepatitis E virus; vaccine