High-Level Production of a Functional Recombinant Hepatitis B Virus Polymerase in Insect Cells with a Baculovirus Expression System

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain ac- tive polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombi- nant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Re- combinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were in- fected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

Expression of Green Fluorescent Protein Gene with Baculovirus Vectorin Insect Cells

The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.

Minerals in edible insects:a review of content and potential for sustainable sourcing

In response to the rapid increase in world population and subsequent demands for food,edible insects represent an alternative food source for humans that is rich in proteins,amino acids and minerals.Entomophagy is a tradition in many countries including China and Thailand,and edible insects have attracted a lot of attention in Western World due to their suitable nutrient composition,high mineral content(e.g.,Fe,Zn,Ca,Mg)and potential use as a supplement in human diet.In this study,we surveyed mineral content in seven insect orders and 67 species of mass produced and wild-harvested edible insects.The total content of essential elements in edible insects was very high in Tenebrio molitor,Bombyx mori,and Zonocerus variegatus.The heavy metal content(summarized for eight species)was below the maximum limit allowed for safe consumption.Sustainable supply of minerals derived from insect biomass is complicated due to the high variations of mineral content in insects and the potential of its change due to processing.

Enhancing forest insect outbreak detection by integrating tree-ring and climate variables

Annual tree rings are widely recognized as valuable tools for quantifying and reconstructing historical forest disturbances.However,the influence of climate can complicate the detection of disturbance signals,leading to limited accuracy in existing methods.In this study,we propose a random under-sampling boosting(RUB)classifier that integrates both tree-ring and climate variables to enhance the detection of forest insect outbreaks.The study focused on 32 sites in Alberta,Canada,which documented insect outbreaks from 1939 to 2010.Through thorough feature engineering,model development,and tenfold cross-validation,multiple machine learning(ML)models were constructed.These models used ring width indices(RWIs)and climate variables within an 11-year window as input features,with outbreak and non-outbreak occurrences as the corresponding output variables.Our results reveal that the RUB model consistently demonstrated superior overall performance and stability,with an accuracy of 88.1%,which surpassed that of the other ML models.In addition,the relative importance of the feature variables followed the order RWIs>mean maximum temperature(Tmax)from May to July>mean total precipita-tion(Pmean)in July>mean minimum temperature(Tmin)in October.More importantly,the dfoliatR(an R package for detecting insect defoliation)and curve intervention detec-tion methods were inferior to the RUB model.Our findings underscore that integrating tree-ring width and climate vari-ables as predictors in machine learning offers a promising avenue for enhancing the accuracy of detecting forest insect outbreaks.

31 Plant Species against Blood Feeding and Disease Vectors Insects: Beyond Anti-Insect Properties, Unvalued Opportunities and Challenges for Health and Sustainability

Plants with bioactive properties are greatly useful in preventing and controlling blood-sucking and disease-vector invertebrates, particularly in developing countries and low-income communities. Their application is a promising alternative to synthetic compounds whose use remains a health, environmental, and economic challenge. However, many are still unknown and unvalued, while others are becoming ignored and threatened. The main objective of this ethnobotanical study is to identify and characterize indigenous and locally grown plants against blood-sucking and disease-vector insects. Salient opportunities and challenges of using these plants are documented and discussed. Semi-structured interviews, using a prepared questionnaire, were conducted with 228 informants. The consensus index (CI) was calculated to analyze the reliability of the collected information. The identified 31 anti-insect plant species belong to 20 botanical families, four morphological categories, and six habitat types. They can be categorized as insecticidal plants (42% of the total), insect repellent (42% of the total), and both insecticidal and insect repellent (16% of the total). More than 54% of these are still abundant in the study area, while about 35.5% have become rare and difficultly accessible. Based on the numerical importance of related anti-insect plant species, the seven targeted blood-sucking insects range in the following decreasing order: Jiggers (16 species) > Fire Ants (9 species) > Flies (8 plants) > Mosquitoes (4 species) > Fleas (2 species) > Bedbugs (1 species) > lice (0 species). The three most commonly used plants, with the highest confirmation indices, are Tetradenia riparia (ICs = 0.712), Eucalyptus globulus subsp. maidenii (ICs = 0.302), and Solanum aculeastrum (ICs = 0.288). The antimicrobial role of many locally grown anti-insect plants and the multiple other associated valorization possibilities are ignored by most informants. Domesticating, propagating, protecting, and promoting the sustainable use of these plants would be an appropriate route for their conservation and continued availability.

Expression of p30, the major surface antigen of Toxoplasma gondii, in baculovirus-insect cell system and the evaluation of immune response induced by p30

Objective: To produce the major surface antigen (p30) of Toxoplasma gondii from the Baculovirus Expression System. Methods: The p30 coding sequence was cloned into a transfer vector, then the recombinant baculovirus containing p30 gene was cloned and purified by the co-transfection and plaque assay. The expression and immunoactivity of the recombinant p30 were analyzed by SDS-PAGE and Western blot. The immune responses in mice for being immunized with recombinant p30 were tested. Results: About 750μg of purified (95% purity) p30 was obtained from a culture of 108 in- sect Sf21 cells. Mice in injected with the recombinant protein produced specific humoral and cellular immune responses. Immunization with p30 also prolonged the period of mice survival infected by Toxoplasma gondii. Conclusion: It is indicated that the recombinant p30 from baculovirus expression system can stimulate mice to produce effective protection from Toxo- plasma gondii infection.

Twenty-Three Years of Insect Pollinator-Dependent Crop Studies in Agro-Ecological Zones of Cameroon (2011-2020)

Cultivation of pollinator-dependent crops has expanded globally, increasing our reliance on insect pollination. This essential ecosystem service is provided by a wide range of managed and wild pollinators whose abundance and diversity are thought to be in decline, threatening sustainable food production. In Cameroon, several studies on pollinator-dependent crops carried out in different agro-ecological zones (AEZ) have been published in national and international journals, in order to present the importance and impact of flowering insects on fruit and seed yields of plant species. We proposed to produce a review article highlighting the different flowering insects and their importance for different plants according to AEZ, without however focusing on the quality of the journal (predator or non-predator) and how the different insects were identified (scientific names given in the publications). Thus, from 1997 to 2020, we collected 116 published papers from which only 26 were kept for this review. The results show that Hymenoptera, including the Apidae, followed by Megachilidae, are the most excellent pollinators of plant species in Cameroon, and they are present in different agro-ecological zones. The majority of publications focused on bees, particularly the honeybee Apis mellifera.

A Review on Characteristics, Extraction Methods and Applications of Renewable Insect Protein

Due to the expected rise in the world population,an increase in the requirements for quality and safety of food and feed is expected,which leads to the growing demand for new sources of sustainable and renewable protein.Insect protein is gaining importance as a renewable material for several reasons,reflecting its potential contribu-tions to sustainability,resource efficiency,and environmental conservation.Some insect species are known to be able to efficiently convert organic waste into high-value products such as protein,requiring less land and water compared to traditional livestock.In addition,insect farming produces fewer greenhouse gas emissions,contri-buting to mitigating climate change.Insects are considered as a major potential alternative to animal or plant protein due to their many nutritional benefits,including high protein,mineral,and vitamin contents.On average,the protein content of insects ranges between 35%and 60%dry weight,which exceeds plant protein sources,such as cereal,soybeans,and lentils.As the acceptance of insect protein grows and technologies advance,the food and feed industries continue to explore and expand their applications,offering consumers diverse and sustainable pro-tein choices.In this review,we discuss the recentfindings relating to insect protein focusing on its characteristics,extraction methods,applications,and opportunities along with some trade-offs and uncertainties.

Social Cost Analysis of Biological Control Technology for Crop Diseases and Insect Pests

With the rapid development of modern agriculture,the prevention and control of crop diseases and insect pests has become an important part to ensure the safety of agricultural production,the quality of agricultural products and the safety of agricultural ecological environment.Although the effect of traditional chemical prevention and control technology is remarkable,the health risks and environmental problems brought by it should not be ignored.As a green and environmentally friendly means of prevention and control,biological prevention and control technology has gradually become a hot research topic and a trend of agricultural production.This paper is intended to comprehensively evaluate the social costs of biological control technologies for crop diseases and pests,including the health risks reduced,environmental improvements,economic benefits,and barriers to promotion,and put forward corresponding policy recommendations.

Phylogenetic Analysis of Baculovirus Isolates from Diseased Insects in Southern Vietnam

The aim of this study was to investigate the molecular identification and assess the genetic relationship of baculovirus isolated from Southern Vietnam. The diseased insect samples were collected from the different fields. The partial sequence of 450 base pairs of lef-8 gene was amplified and sequenced to assess the genetic variations of baculovirus isolates specific for Spodoptera litura, Helicoverpa zea, and Helicoverpa armigera. The sequences alignment demonstrated that Helicoverpa zea specific isolates exhibited six single nucleotide polymorphic sites. Whereas, twenty five single polymorphic sites were found in Spodoptera litura specific isolates. Thus, Spodoptera litura specific isolates were higher polymorphic than Helicoverpa zea specific isolates. The genetic distance analyses showed that the distance between Vietnamese baculovirus isolates and Group II Alphabaculovirus isolates was lower than other Baculovirus groups. The phylogeny of Vietnamese isolates in relation to other baculovirus isolates was also determined using partial sequences of lef-8 gene. The phylogenetic tree placed all Vietnamese isolates in Group II Alphabaculovirus, where seven Vietnamese Helicoverpa zea specific isolates were most closely related to Helicoverpa zea SNPV, fourteen Vietnamese Spodoptera litura specific isolates were located with Spodoptera litura NPV-G2 in one clade and a Vietnamese Helicoverpa armigera isolate was appeared to be closely related to Helicoverpa armigera SNPV-NNg1, Helicoverpa armigera NPV-C1, Helicoverpa armigera NPV-G4.

Analysis of Economic Benefits Brought by Industrialization of Natural Enemy Insects

In the field of biological control of agricultural and forestry pests,natural enemy insects play an important role,constituting the core of modern integrated pest management(IPM)strategy,providing solid support for the zero growth goal of chemical pesticide use,and helping the sustainable development of green agriculture.The rise of the natural enemy insect industry has become a key driver of the transformation of green agriculture,injecting vitality into the sustainability and ecological protection of agriculture.With the increasing concern about food safety,environmental protection and ecological balance all over the world,the application scope of natural enemy insects as biological control means is constantly expanding.Its unique ecological adaptability and environmentally friendly characteristics help to reduce dependence on chemical pesticides,ensure the safety of agricultural products and maintain ecological diversity.The purpose of this study is to analyze the current situation of natural enemy insect industry,evaluate its cost and benefit,emphasize its great significance in promoting the transformation of green agriculture,improving production efficiency and promoting ecological protection by drawing lessons from foreign successful experiences,and explore an efficient and sustainable new agricultural development model.

Sequence and Molecular Evolution Analysis of Ubiquitin Proteins Encoded by Baculoviruses

[Objective] The aim of this study was to analyze the sequence characteristics and molecular evolution of ubiquitins encoded by baculoviruses.[Methods]Clustal W software was used for multiple sequence alignment analysis,and neighbor-joining method（NJ）and maximum parsimony method（MP）were used for the construction of phylogenetic tree.[Results]The baculoviral ubiquitins showed 73%-86% sequence identity to eukaryotic ubiquitin.Two heterogeneous regions of baculoviral ubiquitins were observed：one was the residues from 15-32,the other was located from residues 53 to 60.The else parts were conserved,where many functional amino acids were also observed.Phylogenetic analysis indicated that baculoviral ubiquitins could be divided into three sub-families,including sub-family GV,sub-family I and sub-family II.The molecular evolution of baculoviral ubiquitins might be under negative selection to maintain their functional and structural stability.[Conclusion]The analysis had provided reference for the researches on functional characterization of baculoviral ubiquitins.

Detection of prawn white spot baculovirusby polymerase chain reaction

INTRODUCTIONAprawnbaculovirushasbeenresponsibleformostoftheseriousshrimpdiseaseinChinasince1992.Studiesonthepathology,Pathogenesisandmorphologyofthevirusshowedthatitwasanon-occlUSiontheybaculoviruswhichcouldinfectPenaeusjaponicus,P.nzonham,P.chinests...

Study on purification and ultrastructure of a baculovirus in Penaeus chinensis

A kind of baculovirus was isolated from the cephalothorax homogenate of sick or morbid Penaeus chinensis by differential centrifugation and density gradient centrifugation. Electron microscopic examination of ultrathin section of the gills, stomach and mid-gut tissues also revealed the presence of rod-shaped baculoviral particles with the same size in the affected cell nuclei, where most of the virions arranging in cluster assembled and caused a series of cytopathic changes. The virion covered with bilaminal envelope was 320 ~ 400 nm × 100 ~ 130 nm in size, whereas the nucleocapsid ranged in size of 250~ 300 nm in length and 70 ~ 100 nm in breadth respectively. No nuclear polyhedron or granulin occlusion theies have been found in cells. According to the principle of viral classification, this newly found virus could probably belong to the non-occluded subgroup of insect baculoviridae, i. e., C subgroup baculovirus.

Baculovirus vector-mediated transfer of NIS gene into colon tumor cells for radionuclide therapy

AIM:To investigate the feasibility of radionuclide therapy of colon tumor cells by baculovirus vector-mediated transfer of the sodium/iodide symporter(NIS) gene.METHODS:A recombinant baculovirus plasmid carrying the NIS gene was constructed,and the viruses(BacNIS) were prepared using the Bac-to-Bac system.The infection efficiency in the colon cancer cell line SW1116 of a green fluorescent protein(GFP) expressing baculovirus(Bac-GFP) at different multiplicities of infection(MOI) with various concentrations of sodium butyrate was determined by flow cytometry.An in vitro cytotoxicity assay was also conducted after infection of SW1116 cells with Bac-NIS.Iodine uptake of Bac-NIS infected SW1116 cells and inhibition of this uptake by sodium perchlorate was examined,and the effect of Bac-NISmediated 131 I in killing tumor cells was evaluated by cell colony formation tests.RESULTS:Infection and transgene expression in SW1116with Bac-GFP were significantly enhanced by sodium butyrate,as up to 72% of SW1116 cells were infected with the virus at MOI of 400 and sodium butyrate at 0.5 mmol/L.No obvious cytotoxicity was observed under these conditions.Infection of SW1116 with Bac-NIS allowed uptake of 131 I in these tumor cells,which could be inhibited by sodium perchlorate.The viability of SW1116 cells infected with Bac-NIS was significantly lower than with Bac-GFP,suggesting that NIS gene-mediated 131 I uptake could specifically kill tumor cells.CONCLUSION:Baculovirus vector-mediated NIS gene therapy is a potential approach for treatment of colon cancer.

Interstitial tissue-specific gene expression in mouse testis by intra-tunica albuguineal injection of recombinant baculovirus

The purpose of this study is to establish a gene delivery system for interstitial tissue-specific protein expression in mice testes using modified recombinant baculovirus. Green fluorescent protein （GFP）-expressing recombinant baculovirus （GFP-baculovirus）, in which the insect cell-specific polyhedron promoter was replaced by the cytomegalovirus （CMV）-IE promoter, was used to transfect testicular cells in vitro, and for intra-tunica albuguineal injection of the interstitial tissue of the testis. GFP expression was monitored in frozen testes sections by fluorescence microscopy. Expression of GFP in testicular tissues was also assessed by reverse transcription polymerase chain reaction （RT-PCR）, and protein expression was assessed by Western blot. Testicular cells in vitro were infected efficiently by modified recombinant GFP-baculovirus. lntra-tunica albuguineal injection of GFP- baculovirus into the mouse testis resulted in a high level of GFP expression in the interstitial tissues. RT-PCR analysis clearly showed GFP gene expression in the testis, particularly interstitial tissues. Intra-tunica albuguineal injection of a modified baculovirus that encoded recombinant rat insulin-like growth factor binding protein （IGFBP）-5 resulted in an increase in IGFBP-5 in testis and semen. In conclusion, we have developed an efficient delivery system for gene expression in vivo in testicular cells, particularly cells of the interstitial tissue using intratunica albuguineal injection of a modified recombinant baculovirus. This method will be particularly relevant for application that requires gene delivery and protein expression in the testicular cells of the outer seminiferous tubule of the testis.

Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

Objective: To express human Vascular endothelial growth factor121(VEGF121) in insect cells. Methods: A gene construct containing VEGF was cloned in the p Fast Bac-HTA vector, followed by transformation in DH10 BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by Western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results: Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions: Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

Baculovirus-expressed FAdV-4 penton base protein protects chicken against hepatitis-hydropericardium syndrome

Hepatitis-hydropericardium syndrome(HHS)is an infectious disease caused by fowl adenovirus serotype 4(FAdV-4).Several structural and non-structural proteins of FAdV-4 have been expressed in Escherichia coli and baculovirus expression system to develop candidate subunit vaccines.However,the protective efficiency of baculovirus-expressed penton base protein has not been assessed.In this study,two recombinant capsid proteins,penton base and fiber-2,were constructed.And then,penton base and fiber-2 were administrated alone or together to specific pathogen-free(SPF)chickens at 14 days of life and boosted at 28 days of life.At 42 days of life,the immunized groups and the control group were challenged with FAdV-4 virulent strain.Results show that inoculating penton base or penton base+fiber-2 provided 100%protection to the chickens.All groups vaccinated with the recombinant protein produced detectable antibodies and showed no apparent lesions.Thus,baculovirus-expressed penton base protein is a promising candidate subunit vaccine.

Transgene expression and differentiation of baculovirus-transduced adipose-derived stem cells from dystrophin-utrophin double knock-out mouse

In this study, recombinant baculovirus carrying the microdystrophin and β-catenin genes was used to infect adipose-derived stem cells from a dystrophin-utrophin double knock-out mouse. Results showed that, after baculovirus transgene infection, microdystrophin and β-catenin genes were effectively expressed in adipose-derived stem cells from the dystrophin-utrophin double knock-out mouse. Furthermore, this transgenic expression promoted adipose-derived stem cell differentiation into muscle cells, but inhibited adipogenic differentiation. In addition, protein expression related to the microdystrophin and Wnt/β-catenin signaling pathway was upregulated. Our experimental findings indicate that baculovirus can successfully deliver the microdystrophin and β-catenin genes into adipose-derived stem cells, and the microdystrophin and Wnt/β-catenin signaling pathway plays an important role in myogenesis of adipose-derived stem cells in the dystrophin-utrophin double knock-out mouse.

The Parasitoid Factor in the Virulence and Spread of Lepidopteran Baculoviruses

Insect parasitoids and baculoviruses play important roles in the natural and strategic biological control of insects. The two parasites are frequent competitors within common hosts and much research has focused on the negative impact that baculoviral host infections have on parasitoids. This review summarizes the impacts that parasitoids may have on the virulence and spread of lepidopteran baculoviruses. By changing host behavior and development, parasitoids have been shown to decrease baculovirus virulence and productivity within parasitized baculovirus-susceptible hosts; however, studies of the tools used by hymenopteran parasitoids to overcome their hosts'immune systems, suggest that parasitoids may, in some cases, facilitate baculoviral infections in less susceptible hosts. Laboratory and field research have demonstrated that parasitoids can mechanically transmit baculoviruses between insects, and in this way, increase the efficacy of the viruses. Instances of new, more virulent isolates of baculoviruses have been recorded from specifically parasitoid-targeted hosts suggesting other possible benefits from the transmission or activation of baculoviruses by parasitoids.