title: Generation of virus like particles for epizootic hemorrhagic disease virus.
abstract: Epizootic hemorrhagic disease virus (EHDV) is a distinct species within the genus Orbivirus, within the family Reoviridae. The epizootic hemorrhagic disease virus genome comprises ten segments of linear, double stranded (ds) RNA, which are packaged within each virus particle. The EHDV virion has a three layered capsid-structure, generated by four major viral proteins: VP2 and VP5 (outer capsid layer); VP7 (intermediate, core-surface layer) and VP3 (innermost, sub-core layer). Although EHDV infects cattle sporadically, several outbreaks have recently occurred in this species in five Mediterranean countries, indicating a potential threat to the European cattle industry. EHDV is transmitted by biting midges of the genus Culicoides, which can travel long distances through wind-born movements (particularly over water), increasing the potential for viral spread in new areas/countries. Expression systems to generate self-assembled virus like particles (VLPs) by simultaneous expression of the major capsid-proteins, have been established for several viruses (including bluetongue virus). This study has developed expression systems for production of EHDV VLPs, for use as non-infectious antigens in both vaccinology and serology studies, avoiding the risk of genetic reassortment between vaccine and field strains and facilitating large scale antigen production. Genes encoding the four major-capsid proteins of a field strain of EHDV-6, were isolated and cloned into transfer vectors, to generate two recombinant baculoviruses. The expression of these viral genes was assessed in insect cells by monitoring the presence of specific viral mRNAs and by western blotting. Electron microscopy studies confirmed the formation and purification of assembled VLPs.
authors.affiliation: Dipartimento di Scienze Veterinarie, Università di Pisa, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Forzan
authors.affiliation: The Pirbright Institute, Pirbright, UK.
authors.forename: Sushila
authors.initials: S
authors.lastname: Maan
authors.affiliation: Dipartimento di Scienze Veterinarie, Università di Pisa, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Mazzei
authors.affiliation: The Pirbright Institute, Pirbright, UK.
authors.forename: Manjunatha N
authors.initials: MN
authors.lastname: Belaganahalli
authors.affiliation: Dipartimento di Scienze Veterinarie, Università di Pisa, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Bonuccelli
authors.affiliation: Dipartimento di Biologia, Università di Pisa, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Calamari
authors.affiliation: Scuola Normale Superiore, Pisa, Italy.
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Carrozza
authors.affiliation: Istituto Italiano di Tecnologia, Center for Nanotechnology Innovation, NEST, Pisa, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Cappello
authors.affiliation: NEST, Istituto Nanoscienze-CNR and Scuola Normale Superiore, Pisa, Italy.
authors.forename: Mariagrazia
authors.initials: M
authors.lastname: Di Luca
authors.affiliation: Dipartimento di Scienze Veterinarie, Università di Pisa, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Bandecchi
authors.affiliation: The Pirbright Institute, Pirbright, UK.
authors.forename: Peter P C
authors.initials: PPC
authors.lastname: Mertens
authors.affiliation: Dipartimento di Scienze Veterinarie, Università di Pisa, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Tolari
mesh_terms: D000818:Animals; D000956:Antigens, Viral; D016367:Baculoviridae / Q000235:genetics; D036022:Capsid Proteins / Q000502:physiology*; D002460:Cell Line; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D018164:Hemorrhagic Disease Virus, Epizootic / Q000276:immunology*; D007313:Insecta; D012088:Reoviridae Infections / Q000662:veterinary; D014764:Viral Proteins / Q000235:genetics; D014765:Viral Vaccines / Q000276:immunology; D014771:Virion
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; D014764:Viral Proteins; D014765:Viral Vaccines
keywords: Baculovirus expression system; Epizootic hemorrhagic disease virus; Insect cells; Virus like particles