title: Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli.
abstract: Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein. The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay.
authors.affiliation: CSIRO Manufacturing Flagship, Parkville, Victoria, Australia. Electronic address: lesley.pearce@csiro.au.
authors.forename: Lesley A
authors.initials: LA
authors.lastname: Pearce
authors.affiliation: CSIRO Australian Animal Health Laboratory and Biosecurity Flagship, Geelong, Victoria, Australia.
authors.forename: Meng
authors.initials: M
authors.lastname: Yu
authors.affiliation: CSIRO Manufacturing Flagship, Parkville, Victoria, Australia.
authors.forename: Lynne J
authors.initials: LJ
authors.lastname: Waddington
authors.affiliation: CSIRO Australian Animal Health Laboratory and Biosecurity Flagship, Geelong, Victoria, Australia.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Barr
authors.affiliation: CSIRO Manufacturing Flagship, Parkville, Victoria, Australia.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Scoble
authors.affiliation: CSIRO Australian Animal Health Laboratory and Biosecurity Flagship, Geelong, Victoria, Australia.
authors.forename: Gary S
authors.initials: GS
authors.lastname: Crameri
authors.affiliation: CSIRO Manufacturing Flagship, Parkville, Victoria, Australia.
authors.forename: William J
authors.initials: WJ
authors.lastname: McKinstry
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D045404:Hendra Virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure; D045464:Henipavirus Infections / Q000276:immunology / Q000821:virology; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000648:ultrastructure; D013552:Swine
chemical_list: D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins
keywords: Electron microscopy; Hendra virus; Luminex assay; Nucleocapsid; Purification; Recombinant protein expression