title: Enhanced insecticidal activity of Chilo iridescent virus expressing an insect specific neurotoxin.
abstract: Previously we have generated a recombinant Chilo iridescent virus (CIV) by inserting the green fluorescent protein gene (gfp) into the CIV 157L open reading frame (ORF) locus and showed that this recombinant (rCIV-Δ157L-gfp) was fully infectious both in cell culture as well as in insect larvae. This study opened up a new avenue for increasing the speed of kill of CIV and other iridoviruses by inserting virulence or toxin genes into the viral genome. In the current study we constructed a recombinant CIV (rCIV-Δ157L/gfp-AaIT) where the 157L ORF was replaced with both the AaIT neurotoxin gene from the scorpion Androctonus australis and the gfp gene, each under control of the viral major capsid protein (mcp) gene promoter. Recombinant virus was purified by successive rounds of plaque purification using Spodoptera frugiperda (Sf-9) cells. One-step growth curves for the recombinant viruses, rCIV-Δ157L/gfp-AaIT and rCIV-Δ157L-gfp, and wild-type CIVs in Sf-9 cells showed similar profiles. AaIT toxin expression in infected third instar Galleria mellonella larvae was confirmed by western blot analysis using an antibody against the AaIT protein. rCIV-Δ157L/gfp-AaIT infection at a concentration that kills 100% of the larvae caused paralysis in infected third instar G. mellonella larvae from two days after injection, whereas infection with non-AaIT containing viruses showed mortality starting much later (>10days). Bioassays on these larvae demonstrated that the speed of kill of CIV carrying AaIT was strikingly enhanced as compared to wild-type CIV. These results suggest that insertion of a toxin gene into CIV provides further opportunities to control a wide range of pest insects, such as weevils, using an iridovirus.
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
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: Aydın
authors.initials: A
authors.lastname: Yesilyurt
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: Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: Just M
authors.initials: JM
authors.lastname: Vlak
authors.affiliation: Karadeniz Technical University, Faculty of Science, Department of Biology, 61080 Trabzon, Turkey.
authors.forename: Zihni
authors.initials: Z
authors.lastname: Demirbag
mesh_terms: D000818:Animals; D015153:Blotting, Western; D005818:Genetic Engineering; D005822:Genetic Vectors; D007306:Insecticides; D017994:Iridovirus / Q000235:genetics*; D009036:Moths / Q000821:virology*; D010572:Pest Control, Biological / Q000379:methods*; D016133:Polymerase Chain Reaction; D012604:Scorpion Venoms / Q000235:genetics*
chemical_list: D007306:Insecticides; D012604:Scorpion Venoms; C065634:AaIT neurotoxin, Androctonus australis
keywords: AaIT neurotoxin; Chilo iridescent virus (CIV); Insecticidal activity; Recombinant virus