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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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Articles by Bryony Bonning in JoVE

Other articles by Bryony Bonning on PubMed

Comparative Analysis of the Genomes of Rachiplusia Ou and Autographa Californica Multiple Nucleopolyhedroviruses

The Journal of General Virology. Jul, 2003  |  Pubmed ID: 12810877

The Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV) is a variant of Autographa californica MNPV (AcMNPV) but is significantly more virulent against several major agricultural pests. The genome sequence of the R1 strain of RoMNPV was determined and compared to that of AcMNPV strain C6. The RoMNPV genome is approximately 131.5 kbp with a G+C content of 39.1 %. The homologous repeat regions (hrs) described for AcMNPV-C6 are present in RoMNPV-R1 but the hrs of RoMNPV have fewer palindromic repeats. The RoMNPV-R1 nucleotide sequence is almost completely collinear with the sequence of AcMNPV-C6 and contains homologues of 150 of the 155 ORFs described for AcMNPV-C6. Deletions, insertions and substitutions have resulted in the loss of homologues for AcMNPV ORFs ac2 (bro), ac3 (ctl), ac97, ac121 and ac140 from the RoMNPV genome. The average amino acid sequence identity between RoMNPV and AcMNPV ORFs is 96.1 % and there are differences in promoter motif composition for 23 of these ORFs. Maximum-likelihood analysis of selection pressures on AcMNPV and RoMNPV ORFs indicate that ORFs ro18/ac20-ac21 (arif-1) and ro135/ac143 (odv-e18) have undergone positive selection.

Application of Maximum-likelihood Models to Selection Pressure Analysis of Group I Nucleopolyhedrovirus Genes

The Journal of General Virology. Jan, 2004  |  Pubmed ID: 14718635

Knowledge of virus genes under positive selection pressure can help identify molecular determinants of species-specific virulence or host range without prior knowledge of the mechanisms governing host range and virulence. Towards this end, codon-based models of substitution were used in a maximum-likelihood approach to analyse selection pressures acting on 83 genes of group I nucleopolyhedroviruses (NPVs). Evidence for positive selection was found for nine genes: ac38, ac66, arif-1, lef-7, lef-10, lef-12, odv-e18, odv-e56 and vp80. The baculovirus DNA helicase gene (dnahel) was not found to be positively selected using models that allowed the intensity of selection pressure to vary among codon sites. Further analysis with a method that allows selection pressure intensity to vary among lineages suggests that positive selection may have occurred in dnahel during the divergence of Bombyx mori NPV and the NPVs of Autographa californica and Rachiplusia ou. NPV genes that have undergone positive selection may modulate the ability of different NPVs to replicate efficiently in cells (lef-7, lef-10, lef-12) or to establish primary infection of the midgut (odv-e18, odv-e56) of different host species.

A Simple Wax-embedding Method for Isolation of Aphid Hemolymph for Detection of Luteoviruses in the Hemocoel

Journal of Virological Methods. Mar, 2006  |  Pubmed ID: 16307802

A protocol for isolating hemolymph from viruliferous aphids has been developed. This method uses warm melted wax to immobilize the aphid. Following removal of a hind leg, the hemolymph can be collected readily. Flushing with RNase-free water allows for collection of sufficient hemolymph for RNA extraction from individual aphids. The extracted RNA was successfully used for detection of barley yellow dwarf virus (BYDV) and pea enation mosaic virus (PEMV) from individual viruliferous Rhopalosiphum padi and Acyrthosiphon pisum aphids, respectively. A TaqMan real-time RT-PCR protocol for quantitation of PEMV in the hemolymph of individual aphids was developed. The wax-embedding hemolymph collection technique provides a useful tool for studying molecular interactions between persistent and circulative plant viruses and their insect vectors.

Microbial Control of Black Cutworm (Lepidoptera: Noctuidae) in Turfgrass Using Agrotis Ipsilon Multiple Nucleopolyhedrovirus

Journal of Economic Entomology. Aug, 2006  |  Pubmed ID: 16937664

Agrotis ipsilon multiple nucleopolyhedrovirus (family Baculoviridae, genus Nucleopolyhedrovirus, AgipMNPV), a naturally occurring baculovirus, was found infecting black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), on central Kentucky golf courses. Laboratory, greenhouse, and field studies investigated the potential of AgipMNPV for managing black cutworms in turfgrass. The virus was highly active against first instars (LC50 = 73 occlusion bodies [OBs] per microl with 2-microl dose; 95% confidence intervals, 55-98). First instars that ingested a high lethal dose stopped feeding and died in 3-6 d as early second instars, whereas lethally infected fourth instars continued to feed and grow for 4-9 d until death. Sublethal doses consumed by third or fifth instars had little or no effect on subsequent developmental rate or pupal weight. Horizontal transmission of AgipMNPV in turfgrass plots was shown. Sprayed suspensions of AgipMNPV (5 x 10(8) - 6 x 10(9) OBs/m2) resulted in 75 to > 93% lethal infection of third or fourth instars in field plots of fairway-height creeping bentgrass, Agrostis stolonifera (Huds.), and on a golf course putting green collar. Virus spray residues (7 x 10(9) OBs/m2) allowed to weather on mowed and irrigated creeping bentgrass field plots significantly increased lethal infection of implanted larvae for at least 4 wk. This study, the first to evaluate a virus against a pest in turfgrass, suggests that AgipMNPV has potential as a preventive bioinsecticide targeting early instar black cutworms. Establishing a virus reservoir in the thatch and soil could suppress successive generations of that key pest on golf courses and sport fields.

Virus-derived Genes for Insect-resistant Transgenic Plants

Advances in Virus Research. 2006  |  Pubmed ID: 16997019

Insect viruses have evolved to counter physiological barriers to infection presented by the host insect. For the Lepidoptera (butterflies and moths), these barriers include (1) the peritrophic membrane (PM) lining the gut, which presents a physical barrier to virus infection of the midgut epithelial cells, (2) the basement membrane (BM) that overlies the gut thereby restricting secondary infection of other tissues, and (3) the immune system of the host insect. Hence, insect viruses provide a resource for genes that disrupt host physiology in a specific manner, and these genes in turn serve as a resource both for the study of physiological processes, and for disruption of these processes for pest management purposes. There are several examples of the application of genes used by an insect virus to overcome the PM barrier for production of insect-resistant transgenic plants. There are other examples of intrahemocoelic effectors, such as BM-degrading proteases that can only be used with an appropriate system for delivery of the agent from the gut into the hemocoel (body cavity) of the insect pest. In this chapter, we describe (1) baculovirus- and entomopoxvirus-derived genes that alter the physiology of the host insect, (2) use of these and homologous genes for production of insect-resistant transgenic plants, (3) other viral genes that have potential for use in development of insect-resistant transgenic plants, and (4) the use of plant lectins for delivery of intrahemocoelic toxins from transgenic plants. Plant expression of polydnavirus-derived genes is described by Gill et al. (this volume, pp. 393-426).

A Glassy-winged Sharpshooter Cell Line Supports Replication of Rhopalosiphum Padi Virus (Dicistroviridae)

Journal of Invertebrate Pathology. Feb, 2007  |  Pubmed ID: 17113099

Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.

Localization of a Drosophila Melanogaster Homolog of the Putative Juvenile Hormone Esterase Binding Protein of Manduca Sexta

Insect Biochemistry and Molecular Biology. Feb, 2007  |  Pubmed ID: 17244544

A putative juvenile hormone esterase (JHE) binding protein, P29, was isolated from the tobacco hornworm Manduca sexta [J. Biol. Chem. 275(3), 1802-1806]. A homolog of P29 was identified in Drosophila melanogaster by sequence alignment. This gene, CG3776 was cloned, recombinant DmP29 expressed in Escheriscia coli and two anti-DmP29 antisera raised. In vitro binding of the P29 homolog to Drosophila JHE was confirmed. P29 mRNA and an immunoreactive protein of 25 kDa were detected in Drosophila larvae, pupae and adults. The predicted size of the protein is 30 kDa. Drosophila P29 is predicted to localize to mitochondria (MitoProt; 93% probability) and has a 6 kDa N-terminal targeting sequence. Subcellular organelle fractionation and confocal microscopy of Drosophila S2 cells confirmed that the immunoreactive 25 kDa protein is present in mitochondria but not in the cytosol. Expression of P29 without the predicted N-terminal targeting sequence in High Five cells showed that the N-terminal targeting sequence is shorter than predicted, and that a second, internal mitochondrial targeting signal is also present. An immunoreactive protein of 50 kDa in the hemolymph does not result from alternative splicing of CG3776 but may result from dimerization of P29. The function of P29 in mitochondria and the possible interaction with JHE are discussed.

Impact of a Basement Membrane-degrading Protease on Dissemination and Secondary Infection of Autographa Californica Multiple Nucleopolyhedrovirus in Heliothis Virescens (Fabricus)

The Journal of General Virology. Apr, 2007  |  Pubmed ID: 17374754

ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, that digests components of the basement membrane (BM) during insect metamorphosis. A recombinant baculovirus that expresses ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus and triggers melanization and tissue fragmentation in infected larvae shortly before death. As BMs are a potential barrier to the spread of baculovirus secondary infection to other tissues in the host, this study tested the hypothesis that the rapid death of insects infected with AcMLF9.ScathL was caused by accelerated secondary infection resulting from the degradation of host BMs by ScathL. Viruses expressing catalytically active or inactive ScathL were used to examine the effects of ScathL activity on budded virus release into the haemocoel during infection, the production of polyhedra in infected larvae and the rate of infection of the gut, trachea, haemocytes, fat body and Malpighian tubules. It was concluded that the enhanced insecticidal efficacy of the recombinant baculovirus that expresses ScathL does not result from altered tissue tropism or accelerated systemic infection. Implications for the role of the BM as a barrier to baculovirus dissemination within the host insect are discussed.

Characterisation of Functional and Insecticidal Properties of a Recombinant Cathepsin L-like Proteinase from Flesh Fly (Sarcophaga Peregrina), Which Plays a Role in Differentiation of Imaginal Discs

Insect Biochemistry and Molecular Biology. Jun, 2007  |  Pubmed ID: 17517336

ScathL is a cathepsin L-like cysteine proteinase from Sarcophaga peregrina (flesh fly), which is involved in differentiation of imaginal discs, through proteolysis of components of basement membranes. An expression system based on the methylotrophic yeast Pichia pastoris was used to produce recombinant ScathL. Although the expression construct contained the full proprotein coding sequence for ScathL, the proprotein was only detected in culture supernatant at early stages of expression by Western blotting. The purified recombinant protein contained only a polypeptide similar to mature ScathL, as a result of autocatalytic processing. After activation by reducing agents, the enzyme hydrolysed the cathepsin L substrate Z-Phe-Arg-AMC, with optimal activity at pH 5.5. ScathL showed decreasing activity with increasing ionic strength above 0.3M NaCl, and lost activity irreversibly at pH > or = 7.5. The enzyme showed limited activity towards protein substrates, digesting only to large fragments. ScathL was insecticidal towards larvae of the tomato moth, Lacanobia oleracera, following injection into the haemolymph. It caused melanisation, although no evidence of extensive proteolysis in haemolymph or gut was observed. Production of a inactive mutant form of ScathL showed that enzyme activity was necessary for the complete proprotein processing observed during production as a recombinant protein, and for insecticidal activity.

A Baculovirus-expressed Dicistrovirus That is Infectious to Aphids

Journal of Virology. Sep, 2007  |  Pubmed ID: 17596314

Detailed investigation of virus replication is facilitated by the construction of a full-length infectious clone of the viral genome. To date, this has not been achieved for members of the family Dicistroviridae. Here we demonstrate the construction of a baculovirus that expresses a dicistrovirus that is infectious in its natural host. We inserted a full-length cDNA clone of the genomic RNA of the dicistrovirus Rhopalosiphum padi virus (RhPV) into a baculovirus expression vector. Virus particles containing RhPV RNA accumulated in the nuclei of baculovirus-infected Sf21 cells expressing the recombinant RhPV clone. These virus particles were infectious in R. padi, a ubiquitous aphid vector of major cereal viruses. The recombinant virus was transmitted efficiently between aphids, despite the presence of 119 and 210 vector-derived bases that were stably maintained at the 5' and 3' ends, respectively, of the RhPV genome. The maintenance of such a nonviral sequence was surprising considering that most RNA viruses tolerate few nonviral bases beyond their natural termini. The use of a baculovirus to express a small RNA virus opens avenues for investigating replication of dicistroviruses and may allow large-scale production of these viruses for use as biopesticides.

Potential Ligands of DmP29, a Putative Juvenile Hormone Esterase Binding Protein of Drosophila Melanogaster

Insect Biochemistry and Molecular Biology. Aug, 2007  |  Pubmed ID: 17628282

We previously reported the identification of a putative juvenile hormone esterase (JHE) binding protein DmP29 in Drosophila melanogaster and its primary localization to the mitochondria [Liu, Z., Ho, L., Bonning, B.C., 2007. Localization of a Drosophila melanogaster homolog of the putative juvenile hormone esterase binding protein of Manduca sexta. Insect Biochem. Mol. Biol. 37(2), 155-163]. To further characterize DmP29, we identified potential ligands of this protein. Recombinant DmP29 was shown by ligand blot and co-immunoprecipitation analyses to bind recombinant JHE as well as to larval serum proteins (LSP). The possible biological relevance of the in vitro DmP29-JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolyzed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29-LSP interactions may not be biologically relevant. Given the high abundance, and "sticky" nature of these proteins, interaction of DmP29 with LSP may result from non-specific associations. No DmP29 interactions with non-specific esterases were detected by co-immunoprecipitation analyses. The potential role of DmP29 as a chaperone of JHE is discussed.

Introduction to the Use of Baculoviruses As Biological Insecticides

Methods in Molecular Biology (Clifton, N.J.). 2007  |  Pubmed ID: 17951780

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents. This chapter includes discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy. Formulation and application of baculoviruses for pest control purposes are described elsewhere.

Evaluation of the Insecticidal Efficacy of Wild-type and Recombinant Baculoviruses

Methods in Molecular Biology (Clifton, N.J.). 2007  |  Pubmed ID: 17951782

A considerable amount of work has been done during the last 20 yr to genetically enhance the efficacy of baculovirus insecticides. Following construction of a genetically altered baculovirus, laboratory bioassays are used to quantify various parameters of insecticidal activity such as the median lethal concentration (or dose) required to kill 50% of infected larvae (LC50 or LD50), median survival time of larvae infected at a fixed dose (ST50), and feeding damage incurred by infected larvae. In this chapter, protocols are described for a variety of bioassays and corresponding data analyses for assessment of the insecticidal activity or host range of baculovirus insecticides. Methods are also provided for baculovirus inoculation of larvae using a microapplicator for determining ST50 or for examining physiological effects.

Tissue Specificity of a Baculovirus-expressed, Basement Membrane-degrading Protease in Larvae of Heliothis Virescens

Tissue & Cell. Dec, 2007  |  Pubmed ID: 17959212

ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm Heliothis virescens significantly faster than the wild type virus and triggers melanization and tissue fragmentation shortly before death. The tissue fragmentation was assumed to be a direct consequence of basement membrane degradation by ScathL. The goal of this study was to investigate the tissue specificity of ScathL when expressed by AcMLF9.ScathL using light, transmission and scanning electron microscopy. Baculovirus expression of ScathL resulted in damage to the basement membrane overlying the midgut, fat body and muscle fibers in larvae infected with AcMLF9.ScathL, but not in larvae infected with the control virus AcMLF9.ScathL.C146A or wild type virus AcMNPV C6. Injection of recombinant ScathL and high levels of baculovirus-mediated expression of ScathL resulted in complete loss of the gut. Extensive damage to the basement membrane mediated by ScathL likely resulted in loss of viability of the underlying tissue and subsequent death of the insect. These results confirm the conclusion of an earlier study (Philip, J.M.D., Fitches, E., Harrison, R.L., Bonning, B.C., Gatehouse, J.A., 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Mol. Biol. 37, 589-600) of the remarkable specificity of this protease.

DiPel-selected Ostrinia Nubilalis Larvae Are Not Resistant to Transgenic Corn Expressing Bacillus Thuringiensis Cry1Ab

Journal of Economic Entomology. Dec, 2007  |  Pubmed ID: 18232404

The survival of KS-SC DiPel-resistant and -susceptible European corn borer, Ostrinia nubilalis (Hübner), was evaluated on different tissues from corn, Zea mays L., hybrids, including a nontransgenic and two transgenic corn plants (events MON810 and Bt11) expressing high doses of Bacillus thuringiensis (Bt) Cry1Ab. The survival of Bt-resistant and -susceptible third instars was similar after a 5-d exposure to transgenic plant tissues. Survivors eventually died when returned to Bt corn tissues, but many were able to continue development when transferred to non-Bt corn tissues. Survival of resistant and susceptible larvae also was evaluated in bioassays with dilutions of leaf extracts from the three corn hybrids incorporated in an artificial diet. In these assays, survival was significantly higher for resistant O. nubilalis neonates at three of the five dilutions compared with the susceptible strain, but the resistance ratio was only 2.2- and 2.4-fold for MON810 and Bt11, respectively. The data demonstrate that Bt-resistant and unselected control O. nubilalis larvae were similar in susceptibility to MON810 and Bt11 event corn hybrids. Although we were unable to evaluate the Cry1Ab protein that larvae were exposed to in the transgenic tissue because of company restrictions, Cry1Ab protoxin produced in Escherichia coli was incubated with extracts from non-Bt corn leaves to simulate the in planta effect on the transgenic protein. Cry1Ab protoxin was hydrolyzed rapidly by enzymes in the corn extract into peptide fragments with molecular masses ranging from 132 to 74 kDa, and eventually 58 kDa. Overall, these data suggest that plant enzymes hydrolyze transgenic toxin to one that is functionally activated. Therefore, resistant insect populations with reduced proteinase activity do not seem to pose a threat to the efficacy of commercial MON810 and Bt11 corn hybrids.

Overexpression of Drosophila Juvenile Hormone Esterase Binding Protein Results in Anti-JH Effects and Reduced Pheromone Abundance

General and Comparative Endocrinology. Mar, 2008  |  Pubmed ID: 18275960

The titer of juvenile hormone (JH), which has wide ranging physiological effects in insects, is regulated in part by JH esterase (JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent anti-JH effects. We hypothesize that DmP29 functions in transport of JHE such that over- or under-expression of DmP29 results in increased or decreased JH degradation at specific sites respectively. Overexpression of DmP29 during the first or second instar was lethal, while overexpression during the third instar resulted in eclosion of small adults. Overexpression of DmP29 in newly eclosed flies reduced ovarian development and fecundity in addition to reducing the abundance of aggregation pheromone (cis-vaccenyl acetate) in males and courtship pheromone (cis,cis-7,11-heptacosadiene) in females. Both sexes also had lower levels of 23 and 25 carbon monoenes. Females exhibited reduced receptivity to mating, and males exhibited male-male courtship behavior, with both sexes being hyperactive: Male flies covered 2.7 times the distance of control flies at 2.9 times the maximum velocity. Application of the JH analog methoprene reversed impaired ovarian development, supporting a role for reduced JH in production of this phenotype. Rather than increasing lifespan as expected from a JH deficiency, overexpression of DmP29 reduced the life span of adult flies which may result from the hyperactivity of these flies. Underexpression of DmP29 resulted in reduced longevity, increased fecundity and reduced titers of pupal JHE. An alternative hypothesis, that mitochondrial dysfunction rather than JHE results in the JH-mediated phenotypes, is discussed.

Infectious Genomic RNA of Rhopalosiphum Padi Virus Transcribed in Vitro from a Full-length CDNA Clone

Virology. Jun, 2008  |  Pubmed ID: 18339417

Availability of a cloned genome from which infectious RNA can be transcribed is essential for investigating RNA virus molecular mechanisms. To date, no such clones have been reported for the Dicistroviridae, an emerging family of invertebrate viruses. Previously we demonstrated baculovirus-driven expression of a cloned Rhopalosiphum padi virus (RhPV; Dicistroviridae) genome that was infectious to aphids, and we identified a cell line (GWSS-Z10) from the glassy-winged sharpshooter, that supports RhPV replication. Here we report that RNA transcribed from a full-length cDNA clone is infectious. Transfection of GWSS-Z10 cells with the RhPV transcript resulted in cytopathic effects, ultrastructural changes, and accumulation of progeny virions, consistent with virus infection. Virions from transcript-infected cells were infectious in aphids. This infectious transcript of a cloned RhPV genome provides a valuable tool, and a more tractable system without interference from baculovirus infection, for investigating replication and pathogenesis of dicistroviruses.

Insecticidal Activity of a Basement Membrane-degrading Protease Against Heliothis Virescens (Fabricius) and Acyrthosiphon Pisum (Harris)

Journal of Insect Physiology. May, 2008  |  Pubmed ID: 18395222

ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD(50)) for purified ScathL injected into fifth-instar H. virescens was 11.0 microg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisum with a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 microg/larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity and the potential for use of ScathL in development of insect resistant transgenic plants when combined with an appropriate delivery system.

Baculovirus-expressed Virus-like Particles of Pea Enation Mosaic Virus Vary in Size and Encapsidate Baculovirus MRNAs

Virus Research. Jan, 2009  |  Pubmed ID: 19013202

Pea enation mosaic virus (PEMV: family Luteoviridae) is transmitted in a persistent, circulative manner by aphids. We inserted cDNAs encoding the structural proteins of PEMV, the coat protein (CP) and coat protein-read through domain (CPRT) into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus with and without a histidine tag or an upstream Kozak consensus sequence. The Sf21 cell line provided the highest level of CP expression of the cell lines tested and resulted in production of virus-like particles (VLPs). The CPRT was not detected in recombinant baculovirus-infected cells by Western blot. Addition of a Kozak sequence increased the yield of baculovirus produced VLPs. Baculovirus-expressed VLPs purified on a nickel NTA column were of variable size (13-30 nm) and contained CP mRNA. The purified VLPs were also shown by RT-PCR to contain 70% of 154 baculovirus mRNAs, indicative of non-specific RNA encapsidation in the absence of viral RNA replication. When fed to the pea aphid, Acyrthosiphon pisum (Harris), the VLPs entered the aphid hemocoel, demonstrating that CPRT is not required for uptake of PEMV from the aphid gut. Baculovirus expression of PEMV VLPs provides a useful tool for future analysis of RNA encapsidation requirements and molecular aphid-virus interactions.

The Readthrough Domain of Pea Enation Mosaic Virus Coat Protein is Not Essential for Virus Stability in the Hemolymph of the Pea Aphid

Archives of Virology. 2009  |  Pubmed ID: 19240978

A fraction of the coat protein (CP) subunits in virions of members of the family Luteoviridae contain a C-terminal extension called the readthrough domain (RTD). The RTD is necessary for persistent aphid transmission, but its role is unknown. It has been reported to be required for virion stability in the hemolymph. Here, we tested whether this was the case for pea enation mosaic virus (PEMV) virions in the pea aphid (Acyrthosiphon pisum) using RNA1Delta, a natural deletion mutant lacking the middle portion of the RTD ORF, and CPDeltaRTD, in which the entire RTD ORF was deleted. In infected plants, RNA1Delta virions were as abundant and stable as wild-type (WT) virions, while CPDeltaRTD virions were unstable. No RTD of any size was translated from artificial subgenomic mRNA of CPDeltaRTD or RNA1Delta in vitro. Thus, only the major CP was present in the mutant virions. Using real-time RT-PCR to detect virion RNA, no significant differences in the concentration or stability of WT and RNA1Delta virions were detected in the aphid hemolymph at much longer times than are necessary for virus transmission. Thus, the RTD is not necessary for stability of PEMV RNA in the aphid hemolymph, and it must play another role in aphid transmission.

Toward the Physiological Basis for Increased Agrotis Ipsilon Multiple Nucleopolyhedrovirus Infection Following Feeding of Agrotis Ipsilon Larvae on Transgenic Corn Expressing Cry1Fa2

Journal of Invertebrate Pathology. Oct, 2009  |  Pubmed ID: 19651136

Larvae of the black cutworm, Agrotis ipsilon Hufnagel, were more susceptible to infection by A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV: Baculoviridae) after feeding on Herculex I, a transgenic corn hybrid expressing the Bacillus thuringiensis (Bt)-derived toxin Cry1Fa2 compared to larvae fed on isoline corn. We investigated the physiological basis for increased susceptibility to virus infection following exposure to Herculex I by analyzing the midgut pH, gut protease activity and peritrophic matrix structure which are important factors for both Bt toxin action and baculovirus infection. No significant treatment differences were found in the pH of anterior midgut, central midgut or posterior midgut in larvae fed Herculex I or isoline diets. Analysis of soluble and membrane-associated gut proteinase activities from larvae fed Herculex I or isoline diets indicated that membrane-associated aminopeptidase activity and soluble chymotrypsin-like proteinase activity were significantly lower in Herculex I -fed larvae compared to isoline-fed larvae. The number and relative molecular masses of soluble chymotrypsin-like proteinases did not differ. Baculoviruses were not susceptible to in vitro degradation by bovine chymotrypsin, suggesting that chymotrypsin degradation of baculovirus occlusion-derived virus did not result in reduced infection of larvae fed on isoline diet. Scanning electron micrographs of the peritrophic matrices of Herculex I -fed larvae and isoline-fed larvae indicated that Herculex I did not result in damage to the peritrophic matrix that could facilitate subsequent baculovirus infection. Additional research is required to further delineate the physiological basis for enhanced baculovirus infection following exposure to sublethal doses of Bt toxins.

Dicistroviruses

Annual Review of Entomology. 2010  |  Pubmed ID: 19961327

Dicistroviruses are members of a recently defined and rapidly growing family of picornavirus-like RNA viruses called the Dicistroviridae. Dicistroviruses are pathogenic to beneficial arthropods such as honey bees and shrimp and to insect pests of medical and agricultural importance. Our understanding of these viruses is uneven. We present highly advanced studies of the virus particle structure, remarkable mechanisms of internal ribosome entry in translation of viral RNA, and the use of dicistroviruses to study the insect immune system. However, little is known about dicistrovirus RNA replication mechanisms or gene function, except by comparison with picornaviruses. The recent construction of infectious clones of dicistrovirus genomes may fill these gaps in knowledge. We discuss economically important diseases caused by dicistroviruses. Future research may lead to protection of beneficial arthropods from dicistroviruses and to application of dicistroviruses as biopesticides targeting pestiferous insects.

Autographa Californica Multiple Nucleopolyhedrovirus ODV-E56 Envelope Protein is Required for Oral Infectivity and Can Be Substituted Functionally by Rachiplusia Ou Multiple Nucleopolyhedrovirus ODV-E56

The Journal of General Virology. May, 2010  |  Pubmed ID: 20032203

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. In a previous analysis, the odv-e56 gene was found to be under positive selection pressure, suggesting that it may be a determinant of virus host range. To assess the role of ODV-E56 in oral infectivity and host range, we constructed recombinant AcMNPV clones (Ac69GFP-e56lacZ and AcIEGFP-e56lacZ) in which ODV-E56 protein synthesis was eliminated by inserting a beta-galactosidase (lacZ) expression cassette into the odv-e56 open reading frame. We also constructed a recombinant virus, Ac69GFP-Roe56, in which the native AcMNPV odv-e56 coding sequence was replaced with that of Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV), a closely related virus that is significantly more virulent towards some host species than AcMNPV. The odv-e56 recombinant viruses exhibited no alterations in polyhedron production and morphogenesis or in the production of infectious budded virus in cell culture. In bioassays using three lepidopteran host species, the oral infectivities of the odv-e56 mutant viruses Ac69GFP-e56lacZ and AcIEGFP-e56lacZ were profoundly impaired compared with those of wild-type and control recombinant viruses. Oral infectivity was restored fully by marker rescue of the odv-e56 mutant viruses with either the AcMNPV or the RoMNPV odv-e56 gene. In bioassays using two host species that are more susceptible to RoMNPV than to AcMNPV, Ac69GFP-Roe56 killed larvae with LC50 values similar to those of recombinant viruses expressing AcMNPV ODV-E56. This result indicated that replacement of the AcMNPV odv-e56 gene with the RoMNPV orthologue did not increase virulence against these two species.

A Peptide That Binds the Pea Aphid Gut Impedes Entry of Pea Enation Mosaic Virus into the Aphid Hemocoel

Virology. May, 2010  |  Pubmed ID: 20223498

Development of ways to block virus transmission by aphids could lead to novel and broad-spectrum means of controlling plant viruses. Viruses in the Luteoviridae enhanced are obligately transmitted by aphids in a persistent manner that requires virion accumulation in the aphid hemocoel. To enter the hemocoel, the virion must bind and traverse the aphid gut epithelium. By screening a phage display library, we identified a 12-residue gut binding peptide (GBP3.1) that binds to the midgut and hindgut of the pea aphid Acyrthosiphon pisum. Binding was confirmed by labeling the aphid gut with a GBP3.1-green fluorescent protein fusion. GBP3.1 reduced uptake of Pea enation mosaic virus (Luteoviridae) from the pea aphid gut into the hemocoel. GBP3.1 also bound to the gut epithelia of the green peach aphid and the soybean aphid. These results suggest a novel strategy for inhibiting plant virus transmission by at least three major aphid pest species.

Removal of Transposon Target Sites from the Autographa Californica Multiple Nucleopolyhedrovirus Fp25k Gene Delays, but Does Not Prevent, Accumulation of the Few Polyhedra Phenotype

The Journal of General Virology. Dec, 2010  |  Pubmed ID: 20810745

Low-cost, large-scale production of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) using continuous insect cell culture is seriously hindered by the accumulation of AcMNPV mutants. Specifically, few-polyhedra (FP) mutants, with a reduced yield of occluded virus (polyhedra) and decreased infectivity, usually accumulate upon passaging in cell culture. FP mutations result from transposon insertions in the baculovirus fp25k gene, leading to significantly reduced levels of FP25K protein synthesis. This study evaluated the effects of removing the transposon insertion sites from the wild-type baculovirus fp25k gene; the mutated virus was denoted Ac-FPm. Specifically, this study involved a detailed comparison of wild-type (WT) AcMNPV and Ac-FPm with regard to the proportion of cells having polyhedra, number of polyhedra per cell, the fraction of empty polyhedra, number of occlusion-derived viruses per polyhedron, number of nucleocapsids in the nuclei, FP25K protein synthesis and genetic analysis of the fp25k gene. Removal of TTAA transposon insertion sites from the fp25k gene stabilized FP25K protein synthesis and delayed the appearance of the FP phenotype from passage 5 to passage 10. Electron micrographs revealed that more virus particles were found inside the nuclei of cells infected with Ac-FPm than in the nuclei of cells infected with WT AcMNPV (at passage 10). Abnormalities, however, were observed in envelopment of nucleocapsids and virus particle occlusion within Ac-FPm polyhedra. Thus, the FP phenotype appeared in spite of continued FP25K protein synthesis, suggesting that mechanisms other than fp25k gene disruption can lead to the FP phenotype.

Proteases As Insecticidal Agents

Toxins. May, 2010  |  Pubmed ID: 22069618

Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic metalloproteases and serine proteases have also been examined. The sites of protease toxic activity range from the insect midgut to the hemocoel (body cavity) to the cuticle. This review discusses these insecticidal proteases along with their evaluation and use as potential pesticides.

Next Generation Sequencing Technologies for Insect Virus Discovery

Viruses. Oct, 2011  |  Pubmed ID: 22069519

Insects are commonly infected with multiple viruses including those that cause sublethal, asymptomatic, and latent infections. Traditional methods for virus isolation typically lack the sensitivity required for detection of such viruses that are present at low abundance. In this respect, next generation sequencing technologies have revolutionized methods for the discovery and identification of new viruses from insects. Here we review both traditional and modern methods for virus discovery, and outline analysis of transcriptome and small RNA data for identification of viral sequences. We will introduce methods for de novo assembly of viral sequences, identification of potential viral sequences from BLAST data, and bioinformatics for generating full-length or near full-length viral genome sequences. We will also discuss implications of the ubiquity of viruses in insects and in insect cell lines. All of the methods described in this article can also apply to the discovery of viruses in other organisms.

Autographa Californica Multiple Nucleopolyhedrovirus ODV-E56 is a Per Os Infectivity Factor, but is Not Essential for Binding and Fusion of Occlusion-derived Virus to the Host Midgut

Virology. Jan, 2011  |  Pubmed ID: 20970820

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) envelope protein ODV-E56 is essential for oral infection of larvae of Heliothis virescens. Bioassays with recombinant clones of AcMNPV lacking a functional odv-e56 gene showed that ODV-E56 was required for infectivity of both polyhedra and to a lesser extent, purified ODV. However, binding and fusion assays showed that ODV lacking ODV-E56 bound and fused to midgut cells at levels similar to ODV of wild-type virus. Fluorescence microscopy of midguts from larvae inoculated with ODV-E56-positive and -negative viruses that express GFP indicated that ODV-E56 was required for infection of the midgut epithelium. Purified ODV-E56 bound to several proteins in midgut-derived brush border membrane vesicles, but failed to rescue infectivity of ODV-E56-negative viruses in trans. These results indicate that ODV-E56 is a per os infectivity factor (pif-5) required for primary midgut infection at a point before or after virion binding and fusion.

A Peptide with Similarity to Baculovirus ODV-E66 Binds the Gut Epithelium of Heliothis Virescens and Impedes Infection with Autographa Californica Multiple Nucleopolyhedrovirus

The Journal of General Virology. May, 2011  |  Pubmed ID: 21228132

Baculoviruses infect their lepidopteran hosts via the midgut epithelium through binding of occlusion-derived virus (ODV) and fusion between the virus envelope and microvillar membranes. To identify genes and sequences that are involved in this process, a random phage display library was screened for peptides that bound to brush border membrane vesicles (BBMV) derived from the midgut epithelium of Heliothis virescens. Seventeen peptides that bound to BBMV were recovered. Two of these, HV1 and HV2, had sequence similarity to the ODV envelope protein ODV-E66 that is found in five species of alphabaculoviruses. Chemically synthesized versions of HV1 and HV2, and two peptides (AcE66A and AcE66B) derived from similar sequences of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ODV-E66, bound to unfixed cryosections of whole midgut tissues. AcE66A, but not HV1, bound to H. virescens gut BBMV proteins on a far-Western blot. Competition assays with HV1 and purified AcMNPV ODV resulted in decreased mortality of H. virescens larvae at a dose of 1 LD(50), and a significant increase in survival time at higher virus concentrations. These results suggest a role for ODV-E66 in baculovirus infection of lepidopteran larval midgut epithelium.

Interaction of the Bacillus Thuringiensis Delta Endotoxins Cry1Ac and Cry3Aa with the Gut of the Pea Aphid, Acyrthosiphon Pisum (Harris)

Journal of Invertebrate Pathology. May, 2011  |  Pubmed ID: 21300068

Hemipteran pests including aphids are not particularly susceptible to the effects of insecticidal Cry toxins derived from the bacterium Bacillus thuringiensis. We examined the physiological basis for the relatively low toxicity of Cry1Ac and Cry3Aa against the pea aphid, Acyrthosiphon pisum (Harris). Cry1Ac was efficiently hydrolyzed by aphid stomach membrane associated cysteine proteases (CP) producing a 60kDa mature toxin, whereas Cry3Aa was incompletely processed and partially degraded. Cry1Ac bound to the aphid gut epithelium but showed low aphid toxicity in bioassays. Feeding of aphids on Cry1Ac in the presence or absence of GalNAc, suggested that Cry1Ac gut binding was glycan mediated. In vitro binding of biotinylated-Cry1Ac to gut BBMVs and competition assays using unlabeled Cry1Ac and GalNAc confirmed binding specificity as well as glycan mediation of Cry1Ac binding. Although Cry3Aa binding to the aphid gut membrane was not detected, Cry3Aa bound 25 and 37kDa proteins in aphid gut BBMV in ligand blot analysis and competition assays confirmed the binding specificity of Cry3Aa. This, combined with low toxicity in feeding assays, suggests that Cry3Aa does bind the gut epithelium to some extent. This is the first systematic examination of the physiological basis for the low efficacy of Cry toxins against aphids, and analysis of Cry toxin-aphid gut interaction.

Production of Baculovirus Defective Interfering Particles During Serial Passage is Delayed by Removing Transposon Target Sites in Fp25k

The Journal of General Virology. Feb, 2012  |  Pubmed ID: 21994323

Accumulation of baculovirus defective interfering particle (DIP) and few polyhedra (FP) mutants is a major limitation to continuous large-scale baculovirus production in insect-cell culture. Although overcoming these mutations would result in a cheaper platform for producing baculovirus biopesticides, little is known regarding the mechanism of FP and DIP formation. This issue was addressed by comparing DIP production of wild-type (WT) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with that of a recombinant AcMNPV (denoted Ac-FPm) containing a modified fp25k gene with altered transposon insertion sites that prevented transposon-mediated production of the FP phenotype. In addition to a reduction in the incidence of the FP phenotype, DIP formation was delayed on passaging of Ac-FPm compared with WT AcMNPV. Specifically, the yield of DIP DNA in Ac-FPm was significantly lower than in WT AcMNPV up to passage 16, thereby demonstrating that modifying the transposon insertion sites increases the genomic stability of AcMNPV. A critical component of this investigation was the optimization of a systematic method based on the use of pulsed-field gel electrophoresis (PFGE) to characterize extracellular virus DNA. Specifically, PFGE was used to detect defective genomes, determine defective genome sizes and quantify the amount of defective genome within a heterogeneous genome population of passaged virus.

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