James F. H. Wong

Comparative analysis of the complete genome sequences of Helicoverpa zea and Helicoverpa armigera single-nucleocapsid nucleopolyhedroviruses

The complete nucleotide sequence of Helicoverpa zea single-nucleocapsid nucleopolyhedrovirus (HzSNPV) has been determined (130869 bp) and compared to the nucleotide sequence of Helicoverpa armigera (Ha) SNPV. These two genomes are very similar in their nucleotide (97% identity) and amino acid (99% identity) sequences. The coding regions are much more conserved than the non-coding regions. In HzSNPV/HaSNPV, the 63 open reading frames (ORFs) present in all baculoviruses sequenced so far are much more conserved than other ORFs. HzSNPV has four additional small ORFs compared with HaSNPV, one of these (Hz42) being in a correct transcriptional context. The major differences between HzSNPV and HaSNPV are found in the sequence and organization of the homologous regions (hrs) and the baculovirus repeat ORFs (bro genes). The sequence identity between the HzSNPV and HaSNPV hrs ranges from 90% (hr1) to almost 100% (hr5) and the hrs differ in the presence/absence of one or more type A and/or B repeats. The three HzSNPV bro genes differ significantly from those in HaSNPV and may have been acquired independently in the ancestral past. The sequence data suggest strongly that HzSNPV and HaSNPV are variants of the same virus species, a conclusion that is supported by the physical and biological data.

Pharmacology of insect GABA receptors

A GABA-operated Cl− channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl− channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABAA receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl− channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active att-butylbicy-clophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.

Comparative pathogenesis of Helicoverpa zea S nucleopolyhedrovirus in noctuid larvae

We used a recombinant of Helicoverpa zea S nucleopolyhedrovirus containing the hsp70/lacZ reporter cassette (HzSNPV-hsp70/lacZ) to quantify mortality relationships and to elucidate early pathogenesis in two permissive hosts, Heliothis virescens and Helicoverpa zea, and one semi-permissive host, Trichoplusia ni. Fourth instar T. ni were highly resistant to fatal infection both by oral injection of occlusions and by intrahaemocoelic injection of budded virus, indicating the presence of both midgut and systemic mechanisms of resistance. In bioassays, newly moulted (4(0)) H. zea were significantly more susceptible than 4(0) H. virescens to fatal infection, but mortality levels were the same for larval cohorts inoculated 16 h after the moult (4(16)). Developmental resistance was stronger in H. zea and in both hosts, partially reversed by administration of the optical brightener M2R. In both species, developmental resistance was correlated with a reduced ability of HzSNPV to establish and/or maintain primary midgut infections. In time-course experiments using a dosage of 15 occlusions ( approximately LD(90)), lacZ expression marking the onset of primary and secondary infection was first observed in midgut columnar and tracheal cells at 4 and 12 h, respectively. Inoculation of 4(0) larvae resulted in approximately twofold more foci in H. zea larvae than in H. virescens, but H. zea larvae sloughed infected midgut cells at a faster rate. For both heliothines, interaction of occlusion-derived virus with primary cellular targets within the midgut epithelium was critical to the outcome of infection and a key process underlying acquisition of developmental resistance.

Effect of signal sequence and promoter on the speed of action of a genetically modified Autographa californica nucleopolyhedrovirus expressing the scorpion toxin LqhIT2

Abstract We report on the construction and optimization of recombinant Autographa californica nucleopolyhedrovirus engineered to express the insect-selective toxin IT2 from the scorpion Leiurus quinquestriatus hebraeus . We constructed a series of viruses expressing the synthetic Lqhit2 gene with different signal sequences or controlled by different promoters. The effect of the various viruses on speed of response was assayed in Heliothis virescens larvae. In addition, the performance of the optimum recombinant viral construct was compared with similar constructs carrying the Androctonus australis hector insect toxin controlled by two different promoters. There were no significant differences in speed of response of viruses with the early hr5/ie1 the early hr5/lef3 , or the early/late hr5/39K promoter driving toxin expression. However, the choice of signal sequence resulted in significant effects. The signal sequence from the bombyxin gene of the silkworm Bombyx mori , proved nominally the best. When the signal sequences were used from the following genes, the viruses acted significantly slower: AcMNPV gp67 , a lepidopteran adipokinetic hormone, a dipteran chymotrypsin, and the homologous Lqhit2 . Finally, the signal sequences of the genes for cuticle protein II of Drosophila melanogaster and of the insect toxins of the scorpions A. australis hector and Hottentota judaicus performed very poorly. The speed of action of AcMNPV, carrying the synthetic Lqhit2 gene with the bombyxin secretion signal and driven by the hr5/ie1 promoter [Ac.LqhIT2(hr5/ie1)], was compared to that of the same virus carrying the AaIT gene under the control of the p10 promoter, or the hr5/ie1 promoter in H. virescens , Trichoplusia ni , and Spodoptera exigua larvae. All recombinant viruses elicited the response significantly faster than the common progenitor wild-type virus in all tests. The response elicited by Ac.LqhIT2(hr5/ie1) was nominally faster than that of both viruses expressing AaIT in all insects tested.