Baculovirus Insecticide Production in Insect Larvae.

Baculovirus-based insecticides are currently being used worldwide, and new products are in development in many countries. The most dramatic examples of successful baculovirus insecticides are found in soybean in Brazil and cotton in China. Production of baculoviruses is generally done in larvae of a convenient host species, and the level of sophistication varies tremendously between field-collection of infected insects at the one extreme and automated mass manufacturing at the other. Currently, only products with wild type baculoviruses as active ingredients are commercially available. Baculoviruses encoding insecticidal proteins are considered attractive, especially for crops with little tolerance to feeding damage, where speed-of-kill is an important characteristic. Successful field tests with such recombinant baculoviruses have been done in the past, and more tests are ongoing. However, low-cost production of recombinant baculovirus in larvae poses specific problems, due to the short survival time of the production host.In this chapter, benchtop-scale production of two typical baculoviruses is described. First, we describe the production of wild type Helicoverpa zea nucleopolyhedrovirus in bollworm (H. zea) larvae. H. zea larvae are very aggressive and need to be reared in isolation from each other. Second, we describe the production of a recombinant Autographa californica multiple nucleopolyhedrovirus in the non-cannibalistic cabbage looper, Trichoplusia ni. The recombinant baculovirus encodes the insect-specific scorpion toxin LqhIT2. The tetracycline transactivator system enables the production of wild-type quantity and quality product while toxin expression is repressed since normal toxin production would result in premature death of the production host that would limit progeny virus production.

Baculovirus insecticide production in insect larvae.

Baculovirus-based insecticides are currently being used worldwide, and new products are in development in many countries. The most dramatic examples of successful baculovirus insecticides are found in soybean in Brazil and cotton in China. Production of baculoviruses is generally done in larvae of a convenient host species, and the level of sophistication varies tremendously between field-collection of infected insects at the one extreme and automated mass manufacturing at the other. Currently, only products with wild type baculoviruses as active ingredients are commercially available. Baculoviruses encoding insecticidal proteins are considered attractive, especially for crops with little tolerance to feeding damage, where speed-of-kill is an important characteristic. Successful field tests with such recombinant baculoviruses have been done in the past, and more tests are ongoing. However, low-cost production of recombinant baculovirus in vivo poses specific problems, because of the short survival time of the production host. In this chapter, benchtop-scale production of two typical baculoviruses is described. First, the authors describe the production of wild type Helicoverpa zea nucleopolyhedrovirus in larvae of the bollworm, H. zea. Larvae of this species are very aggressive and need to be reared in isolation from each other. The authors then describe the production of a recombinant Autographa californica multiple nucleopolyhedrovirus in the noncannibalistic cabbage looper, Trichoplusia ni. The recombinant baculovirus encodes the insect-specific scorpion toxin LqhIT2, which severely limits progeny virus production. The tetracycline transactivator system enables the production of wild-type quantity and quality product while toxin expression is repressed.

Production of a recombinant antibody fragment in whole insect larvae.

Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.

Functional characterization of putative promoter elements from infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp and in insect and fish cell lines.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp contains a linear single-stranded DNA genome of approximately 4.1kb with three putative open reading frames (ORFs) on the same DNA strand designated, the Left, Middle, and Right ORFs. The Left ORF codes for non-structural protein and the Right ORF codes for capsid protein, whereas the role of the Middle ORF is still unknown. Two putative promoters, designated P2 and P61, were detected upstream of the Left ORF and Right ORF, respectively. We evaluated the activities of these two promoters with or without a transcriptional enhancer element via the use of firefly luciferase reporter constructs in insect and fish cells, and in shrimp tail muscle. In insect and fish cells, the P2 promoter was stronger than the P61 promoter. The presence of the SV40 enhancer element negatively affected P2 but not P61 promoter activity in insect cells. However, in fish cells, the SV40 enhancer element dramatically increased the activities of both promoters. In shrimp, there was no significant difference in luciferase expression driven by these two promoters. In shrimp tail muscle, the presence of SV40 enhancer element in the construct had no significant effect on the P2 promoter and a negative effect on the P61 promoter. The IHHNV P2 and P61 promoters were found to be constitutive promoters that can drive gene expression in both invertebrate and vertebrate hosts.