title: Replication of Mythimna separata entomopoxvirus in High Five™ cells and the construction of a recombinant.
abstract: Mythimna separata entomopoxvirus (MySEV), of the genus Betaentomopoxvirus, was found to replicate in High Five™ cells. The infected cells produced many occlusion bodies and were hypertrophied but did not lyse. Following infection at a multiplicity of infection of 0.1, titers of extracellular virus reached a plateau 3-4days post infection at 25°C and were estimated at ca. 3×10(5) plaque-forming units per ml in TC-100 or TMN-FH media, both of which contained fetal bovine serum (FBS). Serum free medium, Express Five® SFM, also supported virus replication in High Five™ cells, but the titers were approximately one-tenth of those grown in TC-100 or TMN-FH media containing FBS. Using High Five™ cells, a recombinant MySEV was successfully constructed using homologous recombination. This study opens an avenue to the evaluation of entomopoxvirus gene functions using reverse genetic approaches with in vitro and in vivo hosts.
authors.affiliation: Forestry and Forest Products Research Institute, Matsunosato, Tsukuba, Ibaraki 305-8687, Japan. Electronic address: junsan@ffpri.affrc.go.jp.
authors.forename: Jun
authors.initials: J
authors.lastname: Takatsuka
authors.affiliation: Department of Applied Biological Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Madoka
authors.initials: M
authors.lastname: Nakai
mesh_terms: D000818:Animals; D002460:Cell Line; D017996:Entomopoxvirinae / Q000235:genetics*; D055879:Gene Knock-In Techniques / Q000379:methods*; D005814:Genes, Viral / Q000235:genetics*; D007915:Lepidoptera / Q000821:virology*; D046529:Microscopy, Electron, Transmission; D016133:Polymerase Chain Reaction; D014162:Transfection
keywords: Cell culture; Entomopoxvirus; Mythimna separata; Recombinant virus