Aerosol infectivity of a Baculovirus to Trichoplusia ni larvae: An alternative larval inoculation strategy for recombinant protein production

The baculovirus–insect expression system is a popular tool for recombinant protein production. The standard method for infecting insect larvae with recombinant baculovirus for protein production involves either feeding occlusion bodies or injecting budded virus into the cuticle. In this study, we showed that the recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) at titers >10⁸ pfu/mL efficiently infected Trichoplusia ni (T. ni) larvae through aerosol inoculation of budded virus at a pressure of 5.5 × 10⁴ Pa. The dipping T. ni larvae in virus‐containing solution efficiently infected them. These results indicate that surface contamination, either by aerosol or dipping, lead to infection via spiracles. The aerosol infection route for AcMNPV was restricted to T. ni and Plutella xylostella larvae, whereas Spodoptera litura and Helicoverpa armigera larvae were resistant to this inoculation process. The yields of the reporter proteins DsRed and EGFP from T. ni larvae following aerosol infection were nearly identical to those following oral feeding or injection. This alternative baculovirus infection strategy facilitates recombinant protein and virus production by insect larvae. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Production of Aujeszky's disease (pseudorabies) virus envelope glycoproteins gB and gC as recombinant polyhedra in baculovirus-infected silkworm larvae

The expression of viral antigens in baculovirus-infected insect cells is often ineffective. As an alternative approach, therefore, we developed the recombinant polyhedra technology, which is an efficient strategy for the production of viral subunit vaccine. Here, we report a strategy for the large-scale production of a pseudorabies virus (PRV) gB or gC in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the epitope regions of PRV gB or heparin-binding domains of PRV gC. Recombinant BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae expressed native polyhedrin and fusion protein that was detected using both anti-polyhedrin and anti-PRV gB or anti-PRV-gC antibodies. Electron and confocal microscopy demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin with a normal morphology and that the recombinant polyhedra contained PRV gB or gC. The yield of gB or gC antigen produced in BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae reached 0.69 or 0.46 mg per larva, respectively, at 6 days post-infection. These results demonstrate that the recombinant polyhedra strategy can be used for the large-scale production of PRV gB or gC antigen.     

Dimerization and proper degradation of BmNPV IE2 are required for efficient virus growth in larvae of Bombyx mori (Lepidoptera: Bombycidae)

The baculovirus ie2 gene is one of the immediate early genes, and its product is known to transactivate viral promoters. However, the roles of Bombyx mori nucleopolyhedrovirus (BmNPV) ie2 in insect larvae are poorly understood. Here we investigated the functions of BmNPV IE2 in cultured cells and in insect larvae using two mutant viruses, BmIE2D and BmIE2CS. BmIE2D lacks the IE2 C-terminal coiled-coil domain that is required for IE2 dimerization. The other mutant BmIE2CS expresses an E3 ligase activity-deficient IE2 derivative, which is degraded more slowly compared with wild-type IE2. We found that ie2 mutations had little effect on BmNPV infection in cultured cells, whereas budded virus and occlusion body production was significantly reduced in the hemolymph of B. mori larvae infected with ie2 mutants. These results indicate that both dimerization and proper degradation of BmNPV IE2 are crucial steps for efficient virus growth in B. mori larvae, but not in cultured cells. Oral infection assays also revealed that the infectivity of the occluded form of ie2 mutants was normal in B. mori larvae, which is inconsistent with the results reported from ie2 mutants of Autographa californica NPV. This suggests that loss of IE2 function causes virus-specific effects in host insects.     

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown.In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient.B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.     

Intrinsic antimicrobial properties of silk spun by genetically modified silkworm strains

The domesticated silkworm, Bombyx mori, is a fundamental insect for silk industry. Silk is obtained from cocoons, protective envelopes produced during pupation and composed of single raw silk filaments secreted by the insect silk glands. Currently, silk is used as a textile fibre and to produce new materials for technical and biomedical applications. To enhance the use of both fabrics and silk-based materials, great efforts to obtain silk with antimicrobial properties have been made. In particular, a convincing approach is represented by the enrichment of the textile fibre with antimicrobial peptides, the main effectors of the innate immunity. To this aim, silkworm-based transgenic techniques appear to be cost-effective strategies to obtain cocoons in which antimicrobial peptides are integrated among the silk proteins. Recently, cocoons transgenic for a recombinant silk protein conjugated to the silkworm Cecropin B antimicrobial peptide were obtained and showed enhanced antibacterial properties (Li et al. in Mol Biol Rep 42:19–25,  https://doi.org/10.1007/s11033-014-3735-z, 2015a). In this work we used the piggyBac-mediated germline transformation to generate several transgenic B. mori lines able to overexpress Cecropin B or Moricin antimicrobial peptides at the level of the silk gland. The derived cocoons were characterised by increased antimicrobial properties and the resulting silk fibre was able to inhibit the bacterial growth of the Gram-negative Escherichia coli. Our results suggest that the generation of silkworm overexpressing unconjugated antimicrobial peptides in the silk gland might represent an additional strategy to obtain antimicrobial peptide-enriched silk, for the production of new silk-based materials.     

The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori

The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It’s a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed.     

Screening of candidate proteins interacting with IE-2 of Bombyx mori nucleopolyhedrovirus

IE-2 of Bombyx mori nucleopolyhedrovirus (BmNPV) has been shown to play important roles in baculovirus infection, which are involved in gene expression and viral replication. However, the mechanism remains unknown. In this paper, by TargetP software, four genes, i.e.-2, odv-e26, odv-e56 and BmNPV-gp101 (Ac-orf116) of BmNPV and Autographa californica multiple NPV (AcMNPV) were predicted to be located in mitochondria. By BLAST tool using BmNPV IE-2 protein sequence, 14 NPVs were found to have IE-2 homologues in GenBank, and most of them were predicted to be located in mitochondria, except for that of Antheraea pernyi NPV (AnpeNPV) and Anticarsia gemmatalis NPV (AngeNPV). To observe the subcellular localization of BmNPV IE-2, a recombinant virus overexpressed the IE-2 and eGFP fusion protein was constructed. In infected BmN cells, the fluorescence specifically enriched in the cellular mitochondria. This evidence was accordant with the prediction. Further, Pull-down assay was used to select protein candidates interacting with IE-2 in B. mori cells infected with BmNPV. Of several isolated protein components, sixteen candidates were identified by MALDI-TOF mass-spectrometry, eight baculoviral proteins (ALK-EXO, F protein, IAP-1, LEF-3, LEF-9, ODV-NC42, TLP, and VP39), and eight proteins from B. mori (Actin, ADP/ATP translocase, ATP synthase subunit beta, Beta-tubulin, DNA topoisomerase 2, Histone H4, Soluble guanylyl cyclae alpha-1 subunit, Transketolase). From the functional point of view, most of these proteins were generally divided into two groups, mitochondrial interaction proteins and viral DNA replication proteins. These results implied that the IE-2 had multiple functions involved in regulating viral gene expression, viral replication and also as a component of mitochondrial factors to regulate the cellular energy supply and apoptosis.     

DFMO feeding lowers polyamine levels and causes developmental defects in the silkworm Bombyx mori

The domesticated silkworm Bombyx mori is an economically important insect that produces large quantities of silk during its 5th instar larval stage. Polyamines are important regulators of growth and have been shown to affect silk production, however their role in larval development is not completely understood. L-ornithine decarboxylase (ODC), a key regulatory enzyme in the polyamine biosynthetic pathway catalyzes the conversion of ornithine to putrescine, which is further broken down to spermidine and spermine. In this study, we set out to understand the role of ODC on the growth and development of silkworm larvae. We fed 5th instar larvae with α-difluoromethylornithine (DFMO), an ODC inhibitor and studied its impact on larval silk glands. Feeding DFMO did not alter the expression of L-ODC but led to a significant reduction in putrescine and spermidine levels. Furthermore, reduced cellular levels of polyamine led to increased oxidative stress and decreased cell viability. Subsequently, this resulted in several developmental defects at the pupal and moth stages. These findings highlight the importance of ODC in the growth and development of B. mori larvae.     

High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50–100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.     

Increasing the yield of middle silk gland expression system through transgenic knock-down of endogenous sericin-1

Various genetically modified bioreactor systems have been developed to meet the increasing demands of recombinant proteins. Silk gland of Bombyx mori holds great potential to be a cost-effective bioreactor for commercial-scale production of recombinant proteins. However, the actual yields of proteins obtained from the current silk gland expression systems are too low for the proteins to be dissolved and purified in a large scale. Here, we proposed a strategy that reducing endogenous sericin proteins would increase the expression yield of foreign proteins. Using transgenic RNA interference, we successfully reduced the expression of BmSer1 to 50%. A total 26 transgenic lines expressing Discosoma sp. red fluorescent protein (DsRed) in the middle silk gland (MSG) under the control of BmSer1 promoter were established to analyze the expression of recombinant. qRT-PCR and western blotting showed that in BmSer1 knock-down lines, the expression of DsRed had significantly increased both at mRNA and protein levels. We did an additional analysis of DsRed/BmSer1 distribution in cocoon and effect of DsRed protein accumulation on the silk fiber formation process. This study describes not only a novel method to enhance recombinant protein expression in MSG bioreactor, but also a strategy to optimize other bioreactor systems.     

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.     

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.     

Genetic modification of a baculovirus vector for increased expression in insect cells

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.     

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host–pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection‐driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six‐plex Tandem mass tag (TMT) labeling‐based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post‐infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self‐regulation and response to virus infection. This study provides new insights for understanding the host–virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.     

Advanced technologies for genetically manipulating the silkworm Bombyx mori,a model Lepidopteran insect

Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein- or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed.     

THE MACROMOLECULAR PARACRYSTALLINE LATTICE OF INSECT VIRAL POLYHEDRAL BODIES DEMONSTRATED IN ULTRATHIN SECTIONS EXAMINED IN THE ELECTRON MICROSCOPE

Thin sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus and from silkworm larvae infected with B. mori virus revealed viral particles contained within a pseudohexagonal, macromolecular, paracrystalline lattice. The gipsy moth virus occurs in bundles of one to eight rods enclosed by a limiting membrane. The particles of the silkworm virus, although generally occurring singly, also possess a limiting membrane. The macromolecules appear to be dense, discrete particles when cross-sectioned and to form dense bands by superimposition when longitudinally or obliquely sectioned at certain angles. Calculations of macromolecular size have been made.