title: Production of monoclonal antibodies against the ORF3 protein of rat hepatitis E virus (HEV) and demonstration of the incorporation of the ORF3 protein into enveloped rat HEV particles.
abstract: Eight murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the C-terminal 15-amino-acid portion of the ORF3 protein of rat hepatitis E virus (ratHEV) were produced and characterized. Immunofluorescence assays using the anti-ratHEV ORF3 MAbs revealed the accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmids or inoculated with cell-culture-generated ratHEV strains. Anti-ORF3 MAbs could capture ratHEV particles in culture supernatant and serum following treatment with 0.5 % deoxycholate, but not those without prior detergent treatment or fecal ratHEV particles. Following treatment with 0.5 % deoxycholate and 0.5 % trypsin, the buoyant density of ratHEV particles in culture supernatant with ORF3 protein on the surface shifted from 1.15 g/cm3to 1.26 g/cm3in a sucrose gradient; the resulting particles were capturable by an anti-ORF2 MAb but not by an anti-ORF3 MAb. This indicates that the ORF3 protein (at least its C-terminal portion) is incorporated into the enveloped ratHEV virions released from infected cells but that it is not found in the virions in the feces, supporting the hypothesis that the ratHEV ORF3 protein is associated with the egress of virions from infected cells, similar to human HEV, despite the fact that the ratHEV ORF3 protein lacks a PSAP amino acid motif.
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
authors.forename: Tominari
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
authors.lastname: Tanggis
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
authors.forename: Suljid
authors.initials: S
authors.lastname: Jirintai
authors.affiliation: West Nusa Tenggara Hepatitis Laboratory, Mataram, Indonesia.|Immunobiology Laboratory, Faculty of Medicine, University of Mataram, Mataram, Indonesia.
authors.lastname: Mulyanto
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Nagashima
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nishizawa
authors.affiliation: Center for Experimental Medicine, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kunita
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan. hokamoto@jichi.ac.jp.
authors.forename: Hiroaki
authors.identifier: http://orcid.org/0000-0003-0827-0964
authors.initials: H
authors.lastname: Okamoto
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism; D000914:Antibodies, Viral / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D003593:Cytoplasm / Q000737:chemistry; D003840:Deoxycholic Acid / Q000378:metabolism; D003902:Detergents / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D016752:Hepatitis E virus / Q000737:chemistry* / Q000502:physiology*; D051379:Mice; D051381:Rats; D017422:Sequence Analysis, DNA; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis*; D014771:Virion / Q000737:chemistry / Q000187:drug effects; D019065:Virus Assembly; D057074:Virus Release
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D003902:Detergents; C400973:ORF3 protein, Hepatitis E virus; D014764:Viral Proteins; D003840:Deoxycholic Acid; D014357:Trypsin