Characterization and Cross-transmission of Baculoviruses Infectious to the Diamondback Moth, Plutella xylostella, and some other Lepidopteran Pests of Brassica Crops

Isolates of a granulovirus (GV) from the Diamondback Moth, Plutella xylostella, and nucleopolyhedrovirus (NPV) isolates from Galleria mellonella and Autographa californica were characterized by restriction endonuclease analysis of viral DNA. The capacity for these viruses to infect P. xylostella larvae and some other lepidopteran pests of brassica crops (including Heliothis virescens, Crocidolomia binotalis and Mamestra brassicae) was examined in cross-transmission experiments in which the DNA isolated from purified progeny viruses, was compared by restriction endonuclease analysis with DNA from the inoculum viruses. Two P. xylostella GV isolates from Taiwan and China (Px GV-Taiwan and Px GV-China) appeared to be very closely-related on the basis of comparative restriction endonuclease analysis of viral genomic DNA. However, both virus isolates could be distinguished by 1-3 major band differences and by sub-molar band variation when their DNA was analysed following digestion with Eco RI, Bam HI and Hin dIII. Both P. xylostella GV isolates proved to be infectious for P. xylostella larvae but did not appear to infect M. brassicae, C. binotalis or H. virescens larvae. In contrast, a G. mellonella NPV (Gm NPV) isolate was infectious for P. xylostella larvae as well as for larvae of M. brassicae, C. binotalis and H. virescens. The results also confirmed that P. xylostella larvae are susceptible to infection by A. californica NPV. These studies form the basis for further evaluation of Px GV and Gm NPV as potential biological control agents for the Diamondback Moth.     

Juvenile hormone (JH) esterase activity but not JH epoxide hydrolase activity is downregulated in larval Adoxophyes honmai following nucleopolyhedroviruses infection

Juvenile hormones (JHs) and ecdysteroids are critical insect developmental hormones. JH esterase (JHE) and JH epoxide hydrolase (JHEH) are JH-selective enzymes that metabolize JH and thus regulate the titer of JH. Baculoviruses are known to alter host endocrine regulation. The nucleopolyhedroviruses, AdhoNPV and AdorNPV, are known to have slow and fast killing activity against Adoxophyes honmai (Lepidoptera: Tortricidae), respectively. Here we found that when penultimate (4th) instar A. honmai are inoculated with AdhoNPV or AdorNPV, the mean survival time is 9.7 and 8.2 days, respectively. The larvae molted once but did not pupate. The AdhoNPV- or AdorNPV-infected larvae did not show a dramatic increase in JHE activity as was found in mock-infected larvae, instead they showed a marked decrease in JHE activity. In contrast, both viral infections had no effect on JHEH activity. In order to further characterize the JHE activity, the JHE-coding sequence of A. honmai (ahjhe) was cloned and confirmed to encode a biologically active JHE. Quantitative real-time PCR analysis of ahjhe expression in 4th and 5th instar A. honmai revealed that AdhoNPV and AdorNPV are able to reduce ahjhe expression levels.     

Phylogenetic analysis of <Emphasis Type="Italic">Orgyia pseudotsugata</Emphasis> single-nucleocapsid nucleopolyhedrovirus

The Douglas-fir tussock moth Orgyia pseudotsugata (Lepidoptera: Lymantriidae) is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus (SNPV) isolated from O. pseudotsugata larvae in Canada (OpSNPV) was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group II NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for multiple, concatenated gene phylogenies to classify baculoviruses. Foundation item: Supported by a scholarship from the European Union (Functional Biodiversity and Crop Protection), contract n^o HPMT-CT-2000-00199.     

Entry into midgut epithelial cells is a key step in the selection of genotypes in a nucleopolyhedrovirus

An isolate of the Spodoptera frugiperda multiple nucleopolyhedrovirus comprises a stable proportion of deletion genotypes (e.g., SfNIC-C), that lack pif1 and pif2 rendering them noninfectious per os, and that survive by complementation with a complete genotype (SfNIC-B) in coinfected cells. To determine whether selection for particular ratios of complete and deletion genotypes occurs mainly during the establishment of the primary infection in insect midgut cells or during subsequent systemic infection, we examined genotype frequencies in insects that fed on OBs comprising different co-occluded mixtures of genotypes. Dramatic changes in genotype frequencies were observed between the OB inoculum and budded virus (BV) samples taken from larvae inoculated with OBs comprising 10% SfNIC-B + 90% SfNIC-C indicating that a marked reduction of SfNIC-C genotype had occurred in the insect midgut due to the immediate elimination of all OBs that originated from cells that had been infected only by SfNIC-C. In contrast, immediate changes were not observed in OBs comprising mixtures of 50% SfNIC-B + 50% SfNIC-C or those comprising 10% SfNIC-B + 90% SfNIC-C as most of the OBs in these mixtures originated from cells that had been infected by both genotypes. Subsequent changes in genotypic frequencies during five days of systemic infection were fairly small in magnitude for all genotypic mixtures. We conclude that the prevalence of defective genotypes in the SfNIC population is likely determined by a balance between host selection against OBs produced in cells infected by SfNIC-C alone and within-host selection for fast-replicating deletion genotypes. The strength of intra-host selection is likely modulated by changes in MOI during the infection period.     

Proteomic analysis of nucleopolyhedrovirus infection resistance in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Silkworm hemolymph is an important defense tissue to resist bacteria and virus infections. To study the response of silkworm hemolymph in the resistance of Bombyx mori L. nucleopolyhedrovirus (BmNPV), we constructed a near-isogenic silkworm line with BmNPV resistance using highly resistant and highly susceptible parental strains. In this paper, two-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization (MALDI)-mass spectrometry were employed to investigate the differences of protein patterns in the hemolymph of the highly resistant, highly susceptible and near-isogenic silkworm strains after BmNPV was administrated to the larvae. A comparison between the proteomes of these three silkworm strains led us to identify two differentially expressed proteins, beta-N-acetylglucosaminidase 2 and aminoacylase. The expression levels of these proteins were higher in the BmNPV resistant strains.     

Nucleopolyhedrovirus introduction in Australia

Nucleopolyhedrovirus (NPV) has become an integral part of integrated pest management (IPM) in many Australian agricultural and horticultural crops. This is the culmination of years of work conducted by researchers at the Queensland Department of Primary Industries and Fisheries (QDPI&F) and Ag Biotech Australia Pty Ltd. In the early 1970's researchers at QDPI&F identified and isolated a virus in Helicoverpa armigera populations in the field. This NPV was extensively studied and shown to be highly specific to Helicoverpa and Heliothis species. Further work showed that when used appropriately the virus could be used effectively to manage these insects in crops such as sorghum, cotton, chickpea and sweet corn. A similar virus was first commercially produced in the USA in the 1970's. This product, Elcar(R), was introduced into Australia in the late 1970's by Shell Chemicals with limited success. A major factor contributing to the poor adoption of Elcar was the concurrent enormous success of the synthetic pyrethroids. The importance of integrated pest management was probably also not widely accepted at that time. Gradual development of insect resistance to synthetic pyrethroids and other synthetic insecticides in Australia and the increased awareness of the importance of IPM meant that researchers once again turned their attentions to environmentally friendly pest management tools such NPV and beneficial insects. In the 1990's a company called Rhone-Poulenc registered an NPV for use in Australian sorghum, chickpea and cotton. This product, Gemstar(R), was imported from the USA. In 2000 Ag Biotech Australia established an in-vivo production facility in Australia to produce commercial volumes of a product similar to the imported product. This product was branded, ViVUS(R), and was first registered and sold commercially in Australia in 2003. The initial production of ViVUS used a virus identical to the American product but replicating it in an Australian Helicoverpa species, H. armigera. Subsequent research collaboration between QDPI&F and Ag Biotech reinvigorated interest in the local virus strain. This was purified and the production system adapted to produce it on a commercial scale. This new version of ViVUS, which was branded ViVUS Gold(R), was first registered and sold commercially in 2004. Widespread insect resistance to insecticides and a greater understanding of integrated pest management is leading to increased adoption of technologies such NPV in Australian agriculture.     

The Polyhedral Membrane does not Protect Polyhedra of AcMNPV Against Inactivation on Greenhouse Chrysanthemum

Polyhedral inactivation of wild-type AcMNPV and an AcMNPV mutant lacking the gene for the polyhedral membrane protein (AcMNPV-Delta pp34) was studied on greenhouse chrysanthemum. It was hypothesized that polyhedra without a polyhedral membrane might be more susceptible to inactivation on plants. The density of infectious polyhedra of both viruses on the leaf surface decreased in time in a near-exponential fashion. The inactivation curves suggested the presence of two distinct fractions of polyhedra with differences in persistence. One fraction of polyhedra is not inactivated at all, whereas the other fraction is inactivated in an exponential fashion. Relative inactivation rates of the inactivated polyhedra fraction for wild-type AcMNPV and AcMNPV-Delta pp34 were 0.16 and 0.13 per day, respectively, which is not significantly different. After 28 days on leaves in a greenhouse, both viruses still showed residual infectivity. The fraction of residual infectious polyhedra were not significantly different and amounted to approximately 20% of the original density for both wild-type AcMNPV and AcMNPV-Delta pp34. Therefore, the polyhedral membrane does not protect polyhedra against inactivation on greenhouse chrysanthemum.     

核型多角体病毒侵染及其与宿主免疫系统互作的研究进展

核型多角体病毒(Nucleopolyhedrovirus,NPV)应用广泛,已被开发成微生物杀虫剂和用于重组蛋白表达等.NPV具有两种病毒颗粒:包埋型病毒粒子(occlusion-derived virus,ODV)和芽生型病毒粒子(budded virus,BV),两者的构成和组装存在差异.病毒包涵体在肠道中溶解后释...