A novel recombinant baculovirus expressing insect neurotoxin and producing occlusion bodies that contain Bacillus thuringiensis Cry toxin

A novel recombinant baculovirus, designated AcB5A, was constructed to develop an improved baculovirus insecticide with additional beneficial properties. Bacillus thuringiensis crystal protein gene (cry1–5) and an insect-specific neurotoxin gene (AaIT) were introduced into the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrin–cry1–5–polyhedrin under the control of polyhedrin gene promoter, and by insertion of AaIT under the control of early promoter of ORF3004 from Cotesia plutellae bracovirus. RT-PCR analysis with total RNA from AcB5A-infected cells indicated that cry1–5 and AaIT genes were normally transcribed. The 150 kDa of polyhedrin–Cry1–5–polyhedrin fusion protein was produced by AcB5A and occluded into polyhedra produced by the recombinant virus. This protein was activated when treated with trypsin to form a crystal protein of approximately 65 kDa. The AcB5A showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the lethal time against Spodoptera exigua larvae compared to those infected with wild-type AcMNPV. The expression level of the fusion protein decreased after in vivo passage as a result of homologous recombination between the two polyhedrin genes.     

Insecticidal activity of recombinant baculovirus co-expressing Bacillus thuringiensis crystal protein and Kunitz-type toxin isolated from the venom of bumblebee Bombus ignitus

To improve the insecticidal activity of Autographa californica nucleopolyhedrovirus (AcMNPV), using co-expression of Bacillus thuringiensis crystal protein and a Kunitz-type toxin isolated from bumblebee Bombus ignitus venom, a recombinant AcMNPV, ApPolh5-3006BiKTI, expressing Bi-KTI under the control of early promoter from Cotesia plutellae bracovirus (CpBV) was constructed. In this recombinant virus, B. thuringiensis cry1-5 crystal protein gene was introduced into the genome by the fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. RT-PCR analysis indicated that both Bi-KTI and polyhedrin-cry1-5 fusion protein were successfully expressed from the infected cells. In addition, SDS-PAGE revealed that polyhedrin-cry1-5 fusion protein expressed by recombinant viruses was occluded into the polyhedra. ApPolh5-3006BiKTI showed an improved insecticidal activity against larvae of Plutella xylostella and Spodoptera exigua. At low dosage rates, it was more effective against S. exigua than on P. xylostella, but more rapid insecticidal activity was shown in P. xylostella. These results strongly suggest that co-expression of Bt toxin and Kunitz-type toxins could be successfully applied to improve the insecticidal activity of baculoviruses.     

Co-Occlusion and Persistence of a Baculovirus Mutant Lacking the Polyhedrin Gene

A co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. Coinfection of Spodoptera frugiperda (IPLB-SF-21) tissue culture cells with Autographa californica nuclear polyhedrosis virus (AcMNPV) and an AcMNPV mutant (Ac-E10) lacking the polyhedrin gene resulted in occlusion of both virus types within polyhedra. The amount of occluded Ac-E10 virions in progeny polyhedra populations during serial passage in Trichoplusia ni larvae was evaluated. Maintenance of the mutant in progeny polyhedra required polyhedra inocula containing equal numbers of the two virus types at a high dose. A significant reduction in occluded mutant nucleocapsids occurs with inoculum levels below a 100% lethal dose. At inoculum levels below a 30% lethal dose, the majority of fourth-instar larvae were infected with only one type of virus. The commercial application and ecological advantages of the co-occlusion process are discussed.     

Validation of a Comprehensive Process-Based Model for the Biological Control of Beet Armyworm,Spodoptera exigua,with Baculoviruses in Greenhouses

This paper describes the validation and sensitivity analysis of a process-based simulation model (BACSIM) for the control of beet armyworm, Spodoptera exigua, with baculoviruses. Model predictions are compared to results of independent greenhouse experiments in which second, third, or fourth instar larvae of S. exigua in chrysanthemum plots are treated with different concentrations of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and S. exigua MNPV (SeMNPV), two viruses with distinct differences in infectivity and mean time to kill. BACSIM provides robust predictions for the control of S. exigua populations in greenhouse chrysanthemum with both AcMNPV and SeMNPV. Mortality levels caused by AcMNPV and SeMNPV were generally predicted within a 25% margin of error compared to the observed values. None of the deviations was higher than 40%. All values of simulated foliage consumption, caused by S. exigua populations treated with AcMNPV or SeMNPV applications, fell within 95% confidence intervals of measurements. Simulated time to kill was, in general, lower than the measurements. This discrepancy may be caused by the behavior of S. exigua larvae which feed on the underside of chrysanthemum leaves where they are protected from polyhedra. This suggests that the larval foraging behavior may play an important role in the efficacy of baculovirus applications and should be further studied experimentally. This validated model can be used for the pretrial evaluation of the efficacy of genetically modified baculoviruses as biological control agents and for the optimization of spraying regimes in chrysanthemum cultivation.     

Construction and characterisation of an antifungal recombinant Bacillus thuringiensis with an expanded host spectrum

A novel antifungal Bacillus thuringiensis strain 19–22, ssp. kurstaki (H3a3b3c), was characterised. This strain included cry1Aa, cry1Ab, cry1Ac, and cry1D, which have high insecticidal activities against lepidopteran larvae other than Spodoptera exigua. To expand the host spectrum, a cry1E gene whose product is active against S. exigua was introduced into the isolate. The transformant successfully expressed the Cry1E protein without any loss of its original antifungal activities. These results indicate that this recombinant strain exhibits dual activities and may be used as an integrated control agent to control plant diseases and insect pests.     

Mixed-genotype infections of Trichoplusia ni larvae with Autographa californica multicapsid nucleopolyhedrovirus: Speed of action and persistence of a recombinant in serial passage

Fast-acting recombinant baculoviruses have potential for improved insect pest suppression. However, the ecological impact of using such viruses must be given careful consideration. One strategy for mitigating risks might be simultaneous release of a wild-type baculovirus, so as to facilitate rapid displacement of the recombinant baculovirus by a wild-type. However, at what ratio must the two baculoviruses be released? An optimum release ratio must ensure both fast action, and the eventual competitive displacement of the recombinant virus and fixation of the wild-type baculovirus in the insect population. Here we challenged Trichoplusia ni larvae with different ratios of wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and a derived recombinant, vEGTDEL, which has the endogenous egt gene (coding for ecdysteroid UDP-glucosyltransferase) deleted. Time to death increased with the proportion wild-type virus in the inoculum mixture, although a 1:10 ratio (wild-type: recombinant) resulted in equally rapid insecticidal action as vEGTDEL alone. Five serial passages of three different occlusion body (OB) mixtures of the two viruses were also performed. OBs from 10 larval cadavers were pooled and used to initiate the following passage. Although the wild-type baculovirus was maintained over five passages, it did not go to fixation in most replicates of the serial passage experiment (SPE), and there was no good evidence for selection against the recombinant. Long-term maintenance of a recombinant in serial passage suggests an ecosystem safety risk. We conclude that for assessing ecological impact of recombinant viruses, SPEs in single and multiple larvae are relevant because of potential modulating effects at the between-host level.     

Insecticidal activity of the chitinase from the Spodoptera litura nucleopolyhedrovirus

Baculovirus chitinase gene (chiA) is a late gene essential for liquefying the host insect at a late stage of infection for its hydrolyzing chitin function. In a previous report, baculovirus ChiA has been shown to offer many interesting new opportunities for pest control. Recently, a putative chiA gene was identified in the Korean isolate of the Spodoptera litura nucleopolyhedorvirus (SpliMNPV‐K1) genome. The open reading frame (ORF) contains 1692 nucelotides and encodes a protein of 563 amino acids with a predicted molecular weight of about 62.6 kDa. To study the insecticidal activity of ChiA from SpliMNPV‐K1, we constructed a recombinant AcMNPV, Ap‐SlChiA, which is designed to express the ChiA under the control of a polyhedrin promoter. Western blot analysis indicated that ChiA was successfully expressed by this recombinant virus. Chitinase assay revealed that the chitobiosidase and endochitinase activity of the recombinant virus was 2.5‐ and 3.9‐flods higher than those of wild‐type AcMNPV, respectively. In addition, the recombinant virus showed higher evident insecticidal activity against 3rd instar larvae of Spodotera exigua than that of the AcMNPV. These results suggest that the chiA gene from SpliMNPV‐K1 could be successfully applied to improve pathogenicity of baculoviruses.     

Functional expression of lepidopteran-selective neurotoxin in baculovirus: potential for effective pest management

Recombinant baculovirus expressing insect-selective neurotoxins derived from venomous animals are considered as an attractive alternative to chemical insecticides for efficient insect control agents. Recently we identified and characterized a novel lepidopteran-selective toxin, Buthus tamulus insect-selective toxin (ButaIT), having 37 amino acids and eight half cysteine residues from the venom of the South Indian red scorpion, Mesobuthus tamulus. The synthetic toxin gene containing the ButaIT sequence in frame to the bombyxin signal sequence was engineered into a polyhedrin positive Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the control of the p10 promoter. Toxin expression in the haemolymph of infected larvae of Heliothis virescens and also in an insect cell culture system was confirmed by western blot analysis using antibody raised against the GST-ButaIT fusion protein. The recombinant NPV (ButaIT-NPV) showed enhanced insecticidal activity on the larvae of Heliothis virescens as evidenced by a significant reduction in median survival time (ST50) and also a greater reduction in feeding damage as compared to the wild-type AcMNPV.     

Removal of Adsorbed Toxin Fragments That Modify Bacillus thuringiensis CryIC δ-Endotoxin Iodination and Binding by Sodium Dodecyl Sulfate Treatment and Renaturation

We report that 10- and 25-kDa toxin fragments adhere to CryIC prepared from Bacillus thuringiensis insecticidal crystals, block iodination, and alter membrane binding. There is no apparent affect on CryIC toxicity against Spodoptera exigua. Associated peptides remained bound to CryIC in the presence of 50 mM dithiothreitol or 6 M urea. A novel detergent-renaturation procedure was developed for the purification of B. thuringiensis CryIC toxin. Sodium dodecyl sulfate (SDS) treatment followed by gel filtration chromatography yielded a homogeneous 62-kDa CryIC toxin. After removal of SDS and renaturation, the purified CryIC toxin was fully insecticidal to S. exigua larvae. ¹²⁵I-labeled CryIC bound with high affinity to brush border membrane vesicles from S. exigua larvae.     

Greenhouse Evaluation of Dose– and Time–Mortality Relationships of Two Nucleopolyhedroviruses for the Control of Beet Armyworm, Spodoptera exigua, on Chrysanthemum

Dose– and time–mortality relationships of baculoviruses in pest insects are important for the determination of effective spraying regimes. A series of experiments with Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV) against synchronized populations of S. exigua larvae in greenhouse chrysanthemum was conducted. Dose– and time–mortality relationships of different virus concentrations and S. exigua target stages were determined and the area foliage consumption was measured. Crop injury was greatly reduced when S. exigua were controlled as second or third instar larvae, whereas virus applications against fourth instar larvae could not prevent considerable crop injury, even at high concentrations. SeMNPV was approximately 10 times as infectious as AcMNPV when applied on greenhouse chrysanthemum. The relative virulence of AcMNPV and SeMNPV corresponded reasonably well with previously published laboratory bioassay data. SeMNPV killed second and fourth instar S. exigua larvae approximately 12 h faster than did AcMNPV in chrysanthemum, but no difference in speed of action was found for third instar larvae. The relative speed of action of AcMNPV and SeMNPV determined in chrysanthemum and in laboratory bioassays did not correspond for third instar S. exigua larvae; laboratory bioassay data can therefore not simply be extrapolated to the crop level.     

Development and Field Performance of a Broad-Spectrum Nonviable Asporogenic Recombinant Strain of Bacillus thuringiensis with Greater Potency and UV Resistance

The main problems with Bacillus thuringiensis products for pest control are their often narrow activity spectrum, high sensitivity to UV degradation, and low cost effectiveness (high potency required). We constructed a sporulation-deficient SigK⁻ B. thuringiensis strain that expressed a chimeric cry1C/Ab gene, the product of which had high activity against various lepidopteran pests, including Spodoptera littoralis (Egyptian cotton leaf worm) and Spodoptera exigua (lesser [beet] armyworm), which are not readily controlled by other Cry δ-endotoxins. The SigK⁻ host strain carried the cry1Ac gene, the product of which is highly active against the larvae of the major pests Ostrinia nubilalis (European corn borer) and Heliothis virescens (tobacco budworm). This new strain had greater potency and a broader activity spectrum than the parent strain. The crystals produced by the asporogenic strain remained encapsulated within the cells, which protected them from UV degradation. The cry1C/Ab gene was introduced into the B. thuringiensis host via a site-specific recombination vector so that unwanted DNA was eliminated. Therefore, the final construct contained no sequences of non-B. thuringiensis origin. As the recombinant strain is a mutant blocked at late sporulation, it does not produce viable spores and therefore cannot compete with wild-type B. thuringiensis strains in the environment. It is thus a very safe biopesticide. In field trials, this new recombinant strain protected cabbage and broccoli against a pest complex under natural infestation conditions.     

Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae

The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC₅₀ of Lip and HD1 were 33.27 and 128.61 μg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.     

Construction of a BmNPV polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, Bombyx mori

The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.     

How baculovirus polyhedra fit square pegs into round holes to robustly package viruses

Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross‐braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra‐molecular assembly to specifically entrap massive baculoviruses. Inter‐subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re‐selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano‐particles.     

Use of Proteases to Improve the Insecticidal Activity of Baculoviruses

Basement membranes that surround the tissues of lepidopterous larvae act as potential barriers to baculovirus movement and establishment of systemic infection. Hence, one potential approach to improving the insecticidal activity of baculoviruses is to perforate or eliminate the basement membranes of their hosts, thereby facilitating the process of infection. Toward this end, we constructed six recombinant clones of Autographa californica nucleopolyhedrovirus (AcMNPV) that express three proteases that digest basement membrane proteins: rat stromelysin-1, human gelatinase A, and flesh fly (Sarcophaga peregrina) cathepsin L. Expression of these proteases was directed from either the ie-1 promoter (in AcIE1TV3.STR1, AcIE1TV3.GEL, and AcIE1TV3.ScathL) or the p6.9 promoter (in AcMLF9.STR1, AcMLF9.GEL, and AcMLF9.ScathL). Recombinant proteases were detected in the culture medium of cells infected with recombinant viruses by either zymography or azocoll assay. AcMLF9.STR1 and AcMLF9.ScathL caused premature cuticular melanization of 5th instar Heliothis virescens. Melanization and fragmentation of internal tissues were observed in half of the larvae infected with AcMLF9.ScathL and not at all in larvae infected with AcMLF9.STR1 or wild-type AcMNPV. Lethal-concentration bioassays revealed no significant differences in virulence toward H. virescens among the protease-expressing recombinants and wild-type AcMNPV. However, in survival-time bioassays, AcMLF9.ScathL killed H. virescens approximately 30% faster than AcMLF9.LqhIT2, a virus expressing an insect-selective scorpion neurotoxin from the p6.9 promoter. Larvae infected with AcMLF9.ScathL consumed approximately 26-fold less lettuce than wild-type virus-infected larvae. These results highlight the potential of improving baculovirus efficacy through the expression of proteases.     

The dual-activity insecticidal protein,Cry2Aa,does not enhance the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis

Cry2Aa, one of the major insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki HD1, is known to be active against both lepidopteran and dipteran larvae. In order to determine whether Cry2Aa could enhance or synergize the mosquitocidal activity of B. thuringiensis subsp. israelensis, we constructed a plasmid vector that harbored the cry2Aa operon and transformed crystalliferous and acrystalliferous strains of this bacterium. The wild-type B. thuringiensis subsp. israelensis, a recombinant B. thuringiensis subsp. israelensis producing Cry2A along with its native major mosquitocidal proteins, and a recombinant B. thuringiensis subsp. israelensis producing Cry2Aa alone were tested against three major mosquito species — Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Our results demonstrated that Cry2Aa does not synergize or enhance the mosquitocidal activity of B. thuringiensis subsp. israelensis against these important vectors of disease.     

Complete genome sequence of Bacillus thuringiensis BR145, a strain with insecticidal activity against Lepidoptera pests

Bacillus thuringiensis BR145 isolated from a soybean field in Southern Brazil showed toxicity against two important insect pests from soybean crop, Helicoverpa armigera, and Chrysodeixis includens, with LC₅₀ 0.294 µg.cm^-2 and 0.277 µg.cm^-2, respectively. We analyzed the genome of this strain through sequences obtained by Next Generation DNA Sequencing and de novo assembly. The analysis of the genome revealed insecticidal genes cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Ab, cyt1, and vip3Aa, suggesting the use of this strain in new strategies of biological control.     

Baculovirus‐induced tree‐top disease: how extended is the role of egt as a gene for the extended phenotype?

Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP‐glucosyl transferase (egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree‐top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree‐top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree‐top disease. Here, we studied tree‐top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree‐top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting‐dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree‐top disease in lepidopteran larvae. The conclusion that egt is a ‘gene for an extended phenotype’ is therefore not generally applicable for all baculovirus–host interactions. We hypothesize that in some baculovirus–host systems (including LdMNPV in L. dispar), an effect of egt on tree‐top disease can be observed through indirect effects of egt on moulting‐related climbing behaviour.