title: Construction and characterization of a recombinant invertebrate iridovirus.
abstract: Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-Δ157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol.
authors.affiliation: Karadeniz Technical University, Faculty of Science, Department of Biology, 61080 Trabzon, Turkey.
authors.forename: Arzu
authors.initials: A
authors.lastname: Ozgen
authors.affiliation: Karadeniz Technical University, Faculty of Science, Department of Molecular Biology and Genetics, 61080 Trabzon, Turkey.
authors.forename: Hacer
authors.initials: H
authors.lastname: Muratoglu
authors.affiliation: Karadeniz Technical University, Faculty of Science, Department of Biology, 61080 Trabzon, Turkey.
authors.forename: Zihni
authors.initials: Z
authors.lastname: Demirbag
authors.affiliation: Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Just M
authors.initials: JM
authors.lastname: Vlak
authors.affiliation: Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Monique M
authors.initials: MM
authors.lastname: van Oers
authors.affiliation: Karadeniz Technical University, Faculty of Science, Department of Biology, 61080 Trabzon, Turkey. Electronic address: remziye@ktu.edu.tr.
authors.forename: Remziye
authors.initials: R
authors.lastname: Nalcacioglu
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D059765:Homologous Recombination; D017994:Iridovirus / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification / Q000502:physiology; D013194:Staining and Labeling; D010948:Viral Plaque Assay; D048889:Weevils
chemical_list: D049452:Green Fluorescent Proteins
keywords: 157L; Apoptosis; Chilo iridescent virus; Green fluorescent protein; Recombinant virus