title: Generation of menangle virus nucleocapsid-like particles in yeast Saccharomyces cerevisiae.
abstract: Menangle virus (MenV), which was isolated in Australia in 1997 during an outbreak of severe reproductive disease in pigs, is a novel member of the genus Rubulavirus in the family Paramyxoviridae. Although successfully eradicated from the affected piggery, fruit bats are considered to be the natural reservoir of the virus and therefore an ongoing risk of re-introduction to the pig population exists. Accordingly, reagents to facilitate serological surveillance are required to enhance the diagnostic capability for MenV, which is a newly recognized cause of disease in pigs with the potential to severely affect production in naive breeding herds. To address this need, recombinant MenV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. Using the expression vector pFGG3 under control of the GAL7 promoter, high yields of recombinant MenV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology, although not in length, to authentic nucleocapsids from virus-infected cells. Electron microscopy analysis also showed that yeast-expressed N protein which lacked the C-terminal tail (amino acid residues 400-519) formed significantly longer and denser nucleocapsid-like particles. Nucleocapsid-like particles derived from the full-length recombinant protein were stable and readily purified by CsCl gradient ultracentrifugation. When used as coating antigen in an indirect ELISA, the recombinant N protein reacted with sera derived from pigs experimentally infected with MenV and a simple serological assay to detect MenV-specific antibodies in pigs, fruit bats and humans could be designed on this basis.
authors.affiliation: Institute of Biotechnology, V. Graiciuno 8, Vilnius LT-02241, Lithuania.
authors.forename: Mindaugas
authors.initials: M
authors.lastname: Juozapaitis
authors.forename: Andrius
authors.initials: A
authors.lastname: Serva
authors.forename: Indre
authors.initials: I
authors.lastname: Kucinskaite
authors.forename: Aurelija
authors.initials: A
authors.lastname: Zvirbliene
authors.forename: Rimantas
authors.initials: R
authors.lastname: Slibinskas
authors.forename: Juozas
authors.initials: J
authors.lastname: Staniulis
authors.forename: Kestutis
authors.initials: K
authors.lastname: Sasnauskas
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Shiell
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Bowden
authors.forename: Wojtek P
authors.initials: WP
authors.lastname: Michalski
mesh_terms: D010252:Paramyxoviridae / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D014771:Virion / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins