title: Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.
abstract: Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.
authors.affiliation: Centro de Investigación en Sanidad Animal (CISA), Instituto Nacional de Investigación y Tecnología Agraria y Almentaria (INIA), Ctra de Valdeolmos-El Casar s/n, Valdeolmos, 28130, Madrid, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Marín-López
authors.affiliation: Centro de Investigación en Sanidad Animal (CISA), Instituto Nacional de Investigación y Tecnología Agraria y Almentaria (INIA), Ctra de Valdeolmos-El Casar s/n, Valdeolmos, 28130, Madrid, Spain. ortego@inia.es.
authors.forename: Javier
authors.initials: J
authors.lastname: Ortego
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D001820:Bluetongue virus / Q000235:genetics* / Q000276:immunology; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors; D012756:Sheep / Q000276:immunology / Q000821:virology; D014611:Vaccination / Q000379:methods* / Q000662:veterinary; D014614:Vaccines, Synthetic / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014616:Vaccinia virus / Q000235:genetics* / Q000276:immunology; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; C078966:NS1 protein, bluetongue virus; C442520:VP2 protein, Bluetongue virus; D014614:Vaccines, Synthetic; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C066862:VP7 protein, orbivirus
keywords: Bluetongue virus; NS1 and VP7 proteins; Recombinant modified vaccinia virus Ankara; VP2; Viral-vectored vaccine