Downloaded from www.microbiologyresearch.org by IP: 23.22.50.124 On: Wed, 23 Mar 2016 06:45:01 Journal of General Virology (1995), 76, 2447-2456. Printed in GreatBritain 2447 Characterization, sequencing and phylogeny of the ecdysteroid UDP-glucosyltransferase gene from two distinct nuclear polyhedrosis viruses isolated from Choristoneurafumiferana John W. Barrett, la Peter J. KrelF and Basil M. Arif l* 1Natural Resources Canada, Canadian Forest Service, Sault Ste. Marie, Ontario P6A 5M7 and 2Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2 W1, Canada The ecdysteroid UDP-glucosyltransferase (ego gene isolated from a plaque-purified isolate of Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus (CfMNPV) was compared to its homologue from a defective MNPV virus (CfDEF) present in wild-type virus populations infecting the eastern spruce budworm, C. fumiferana. The egt genes were located in the same relative position within the virus genomes and their genomic location and arrangement were similar to that found in Autographa californica MNPV (AcMNPV) and Orgyia pseudotsugata MNPV (OpMNPV). The genes encoded 491 and 494 amino acid open reading frames respectively, and were 67 % identical at the amino acid level and 74 % identical at the nucleotide level. Tran- scripts of the egt of CfMNPV peaked around 12 h post- infection (p.i.) and disappeared after 36 h p.i. Tran- scripts of the egt of CfDEF peaked between 6 and 9 h p.i. and were not detected 24 h p.i. The egt from CfMNP¥ was more similar to the partially sequenced egt identified from OpMNPV, at the nucleotide and amino acid levels, than it was to the egt from the CfDEF, AcMNPV, Bombyx mori NPV, Lymantria dispar MNPV or Spodop- tera exigua MNPV. Phylogenetic analysis of egt sup- ported the baculovirus evolution scheme suggested by polyhedrin sequence analysis. Introduction In insects, the moulting hormone ecdysone initiates and regulates a cascade of genes expressed during moulting. Recent studies have revealed that baculoviruses have evolved mechanisms to delay normal lepidopteran larval moulting. The ecdysteroid UDP-glucosyltransferase (egt) gene, first identified in Autographa californica multi- nucleocapsid nuclear polyhedrosis virus (AcMNPV), catalyses the conjugation of ecdysone and UDP-glucose (O'Reilly & Miller, 1989), and results in the clearing of ecdysone from the haemolymph. The direct result is a delay in moulting so that virus-infected larvae feed longer and cause greater foliar damage. Interruption of the AcMNPV egt coding region increased the virulence, but did not reduce the infectivity of the virus. Insects infected with the modified AcMNPV egt virus fed less and died more rapidly than those infected with unaltered * Author for correspondence. Fax + 1 705 759 5700. e-mail barif@am.ncr.forestry.ca The sequence data reported here will appear in the GenBank Database under the accessionnumbers U10441 and U10476, AcMNPV (O'Reilly & Miller, 1991). These advantages suggested that alteration in the expression of egt in other baculoviruses, either through deletion or inactivation, might also provide a more effective biological control insecticide. The eastern spruce budworm, Choristoneura fumi- ferana, is an economically important forest pest and is responsible for the destruction of approximately 10"1 million hectares of forest per year in Canada (Anon- ymous, 1994). To date, there is no effective and economically viable biological control agent against this devastating pest. At present, formulated Bacillus thu- ringiensis (B.t.) is used to control forest lepidopteran pest species. However B.t. has the potential to impact on non- target lepidopteran species and has short persistence times (Cunningham & van Frankenhuyzen, 1991). Baculoviruses are naturally occurring pathogens which are generally insect specific. It has been demonstrated that genetic modification of baculoviruses can improve their effectiveness as pest control agents (Wood & Granados, 1991). Manipulation of the CfMNPV egt appears to be one viable approach to improve baculo- viruses against the spruce budworm. We report the identification and characterization of the egt genes 0001-3271 © 1995SGM