title: Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces.
abstract: Ten murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the well-conserved, C-terminal 24-amino acid portion of ORF3 protein of hepatitis E virus (HEV) were produced and characterized. Immunofluorescent assays using the anti-ORF3 MAbs revealed accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmid or inoculated with cell-culture-generated HEV. The anti-ORF3 MAbs could capture HEV particles in culture medium and serum at variable efficiency of up to 61 and 49%, respectively, but not those in feces. By sandwiching between immobilized and enzyme-labeled anti-ORF3 MAbs in ELISA, ORF3 antigen was detected in the culture media with an HEV RNA titer of >10(6) copies/ml and increased in parallel with the increase in HEV load. HEV progenies in the culture supernatant, with ORF3 protein on the surface, banded at a low buoyant density of 1.15 g/cm(3) in sucrose. A representative anti-ORF3 MAb (TA0536) could partially neutralize the infection of cell-culture-generated HEV in a cell culture system. These results indicate that ORF3 protein, at least its C-terminal portion, is present on the surface of HEV virions released from infected cells and support a previously proposed assumption that ORF3 protein is associated with virus release from infected cells.
authors.affiliation: Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-Shi, Tochigi-Ken 329-0498, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takahashi
authors.forename: Kentaro
authors.initials: K
authors.lastname: Yamada
authors.forename: Yu
authors.initials: Y
authors.lastname: Hoshino
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takahashi
authors.forename: Koji
authors.initials: K
authors.lastname: Ichiyama
authors.forename: Toshinori
authors.initials: T
authors.lastname: Tanaka
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Okamoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D045744:Cell Line, Tumor; D003470:Culture Media / Q000737:chemistry; D005243:Feces / Q000821:virology*; D016751:Hepatitis E / Q000175:diagnosis / Q000276:immunology*; D016752:Hepatitis E virus / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016366:Open Reading Frames; D016415:Sequence Alignment; D044967:Serum / Q000276:immunology / Q000821:virology*; D014764:Viral Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D003470:Culture Media; D014764:Viral Proteins