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PubMed2024

Characterization of West Nile virus Koutango lineage from phlebotomine sandflies in Kenya.

The West Nile virus (WNV), primarily transmitted by mosquitoes, is one of the most widespread flaviviruses globally, with past outbreaks occurring in the USA and Europe. Recent studies in parts of Africa, including Kenya, have identified the West Nile virus Koutango lineage (WN-KOUTV) among phlebotomine sandfly populations, however, our understanding of this virus remains limited. This study aimed to characterize WN-KOUTV from phlebotomine sandflies. Sandflies were sampled between 12th -16th March 2021 and 16th -20th March 2023 from six villages each in Baringo and Isiolo Counties, using CDC light traps. Female sandflies were taxonomically identified and pooled based on genus and site of collection. Virus isolation was performed in Vero cells. Viral genomes were determined using next-generation sequencing. Phylogenetic and molecular clock analyses were done to decipher the virus's evolutionary relationships. Comparative analyses of amino acid sequences were performed to determine variations. Protein modeling in Pymol was conducted to elucidate variations in key protein regions. Evolutionary pressure analysis investigated the selection pressures on the virus. In vitro experiments were done to investigate the virus growth kinetics in mammalian Vero E6 and mosquito C6/36 cells. We report the isolation of WN-KOUTV from Salabani in Baringo and Aremet in Isiolo, Kenya. The isolated WN-KOUTVs clustered with previously identified WN-KOUTV strains. Comparative analysis revealed a unique amino acid at NS5 653. The WN-KOUTV lineage as a whole is under purifying selective pressure, with diversifying pressure acting at site NS3 267. The current WN-KOUTV replicated in Vero E6 and C6/36 cells comparable to West Nile virus Lineage 1a, isolated from mosquitoes. Subsequent isolations of WN-KOUTV in phlebotomine sandflies suggest potential vectors, however, vector competence studies would confirm this. Replication in mammalian and insect cell lines suggests there may exist a vector/host relationship. We speculate the close genetic relationship of WN-KOUTV strains from East and West Africa may potentially be enabled by bird migratory routes between the two regions. If proven, this could point to a potential future pandemic pathway for this virus.

Animals↗

PubMed2005

Tenebrio molitor antifreeze protein gene identification and regulation.

The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

Amino Acid Sequence↗

PubMed1994

Vaccinia virion surface polypeptide Ag35 expressed from a baculovirus vector is targeted to analogous poxvirus and insect virus components.

Polypeptide Ag35, a major early component of the vaccinia surface, is integrated into the formative viral lipoprotein tegument. To ascertain whether positioning of Ag35 is due to its general affinity for newly assembled viral membranes we created a recombinant A12 vector to express the vaccinia protein. The baculovirus system was chosen because intranuclear virions of this agent are likewise enclosed inside newly formed envelopes. Comparable infections of two insect cell lines established that more abundant synthesis occurred in High Five (H5) than in SF9 cells. We, therefore, used H5 cells for most experiments reported here. Combined analyses by PAGE, Western blotting, and immunocytology, using light and electron microscopy, revealed a dissemination of Ag35 throughout the cell. Higher concentrations were evident at the cell surface, nuclear perimeter, and within intranuclear virogenic stroma. The association with the virogenic stroma was of specific interest with respect to vaccinia development because it showed a similarity in the targeting of Ag35 toward intranuclear DNA-protein foci of baculovirus which are analogous to the vaccinia-specified cytoplasmic "factories." A further remarkable analogy concerns association of Ag35 with intranuclear baculovirus envelopes, revealing a propensity of Ag35 for nascent viral lipoprotein membranes.

Animals↗

PubMed2003

Functional reverse transcriptase encoded by the human LINE-1 from baculovirus-infected insect cells.

The human LINE-1 ORF2, which encodes reverse transcriptase, was inserted into a baculovirus shuttle vector and expressed in Sf 21 cells. An immunoreactive polypeptide (149kDa) synthesized by infected cells had reverse transcriptase activity. A procedure for purification of functional ORF2 protein from insect cells was developed. The enzyme was purified with good recovery to near homogeneity and retained stable DNA polymerase activity. The optimum reaction conditions of the enzyme were determined with respect to salts, pH, and temperature. Substrate specificities and divalent cation requirements were investigated. The recombinant enzyme had a 3-fold preference for Mg2+ over Mn2+ for reverse transcriptase activity on poly(rA).oligo(dT)(12). As for DNA synthesis, the recombinant ORF2 protein was found to possess both RNA-dependent and DNA-dependent DNA polymerase activities.

Animals↗

PubMed1988

Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector.

The human insulin receptor (IR) is a transmembrane glycoprotein, whose cytoplasmic domain contains an insulin-activated protein-tyrosine kinase (EC 2.7.1.112). By the use of an appropriately engineered baculovirus expression vector, a soluble cytoplasmic derivative of this domain was expressed in the insect cell line Spodoptera frugiperda (Sf9). At 24 to 48 h after Sf9 cells were infected with recombinant virus, a protein of the size expected for this domain (approximately 48 kilodaltons) constituted a major band when total cell lysates of metabolically labeled cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. This protein (designated AchIRPTK) was immunoprecipitated by three monoclonal antibodies, each of which recognizes a distinct antigenic site of the IR cytoplasmic domain and requires the native structure of the protein for recognition and one of which binds at or near the physiologically relevant site(s) of IR autophosphorylation. In vivo, AchIRPTK was phosphorylated on both tyrosine and serine residues. When affinity purified, the kinase was active in vitro; it autophosphorylated exclusively on tyrosine residues, and phosphorylated the exogenous substrates histone H2b and poly(Glu-Tyr). The expression of an active IR protein-tyrosine kinase molecule in this heterologous cell system provides an efficient experimental method for producing this domain in quantity for enzymatic and structural studies.

Animals↗

PubMed2006

Protein-DNA interactions in the promoter region of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide of the cotton bollworm, Helicoverpa armigera.

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are two crucial neuropeptides which regulate insect development and sex pheromone biosynthesis respectively. These peptides are encoded by a single gene, termed DH-PBAN gene. In this study, we characterized the promoter of the DH-PBAN gene in Helicoverpa armigera (Har). Transient transfection assays using a series of stepwise deletion fragments linked to the luciferase reporter gene indicate that the promoter contains multiple regulator domains that can activate and repress reporter gene expression. The fragment spanning -467 to -371 bp of the DH-PBAN promoter is an activator domain of transcription, whereas the region from -965 to -534 bp represses the promoter activity in the insect cell line BmN. Electrophoretic mobility shift assays demonstrate that at least two nuclear protein factors from the nuclear protein extracts of H. armigera suboesophageal ganglion, Har-DHMBP-1 and-2 (DH-modulator-binding protein) can specifically bind to the activating region. Furthermore, we characterized in detail that the nuclear protein factor Har-DHMBP-3 can specifically bind to a classical E-box, CAGCTG localized at positions -360 to -355 bp, a potential site for interaction with basic helix-loop-helix transcription factors. Mutation of this E-box results in a significant reduction of the promoter activity, suggesting it can modulate the previously identified activator domain. Taken together, multipartite cis-elements and transcription factors in the DH-PBAN promoter are involved in regulation of the gene expression.

Animals↗

PubMed1997

Comparison of the response of midgut epithelial cells and cell lines from lepidopteran larvae to CryIA toxins from Bacillus thuringiensis.

The cytotoxic responses of midgut epithelial cells (MEC) from spruce budworm (SBW), gypsy moth (GM) and silkworm (SW) larvae were compared with the cytotoxic response of lepidopteran cell lines (SF-9, SE-1a, and CF-1) to CryIA toxins from Bacillus thuringiensis. The MEC from SBW, SW and GM had binding proteins for CryIA(a,b,c) toxins, whereas the lepidopteran cell lines had binding proteins for CryIA(c). Single MEC exposed to CryIA(a,b,c) toxins in a qualitative lawn assay were equally susceptible to the toxins with a threshold response at about 1ng. The cell lines were not susceptible to CryIA(a,b) toxins in the dose range tested, but had threshold responses for CryIA(c) of 3.4ng for SF-9, 50.2ng for SE-1a and 5.9ng for CF-1. In the quantitative Live/Dead assay, MEC were equally susceptible to CryIA(a,b,c) toxins with a threshold effect at about 1ng and a maximum effect at about 10ng. CF-1 was most sensitive to CryIA(c) with a threshold effect at 0.39ng and a maximal effect at about 1ng. In contrast, a 25-50 times greater dose of CryIA(a) or CryIA(b) was required to elicit a similar response as CryIA(c) for CF-1. SF-9 and SE-1a were most susceptible to CryIA(c) with a threshold effect observed at about 0.5ng and maximal effects at about 2ng. SF-9 cells have a threshold and maximum response to CryIA(a,b) of about 10ng and 20ng, respectively. SE-1a cells have a threshold and maximal response to CryIA(a,b) of 5ng and 10ng, respectively. Intact midgut epithelium exposed to CryIA(a,b,c) toxins had a threshold dose of 2ng for CryIA(b), 10-30ng for CryIA(a) and 2-30ng for CryIA(c). This study has shown that MEC are affected by a broader spectrum of toxins compared to the lepidopteran larvae and insect cell lines.

Journal Article↗

PubMed2001

Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system.

Human secreted alkaline phosphatase (SEAP) was produced in a stably-transformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.

Alkaline Phosphatase↗

PubMed1998

Synthesis of rotavirus-like particles in insect cells: comparative and quantitative analysis.

When the three major structural proteins, VP2, VP6, and VP7, of rotavirus are co-expressed in insect cells infected with recombinant baculoviruses, they self-assemble into triple-layered virus-like particles (VLPs) that are similar in morphology to native rotavirus. In order to establish the most favorable conditions for the synthesis of rotavirus VLPs, we have compared the kinetics of 2/6/7-VLP synthesis in two different insect cell lines: High Five cells propagated in Excell 405 medium and Spodoptera frugiperda 9 cells in Excell 400 medium. The majority of VLPs produced in both cell lines were released into the culture medium, and these released VLPs were predominantly triple-layered and were found to be stable for the period of six or seven days examined. The optimal synthesis of VLPs depended upon the cell line and the culture medium used as well as the time of harvesting infected cell cultures. The highest yield of VLPs was obtained from High Five cultures in the late phase of infection when the yield was at least 5-fold higher than that from S. frugiperda 9 cultures on a per cell basis. Our results demonstrate the usefulness of High Five cells for the production of VLPs as potential rotavirus subunit vaccines.

Animals↗

PubMed1999

Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.

Animals↗

PubMed1997

Effect of demethylation on the chitinase inhibitory activity of allosamidin.

Removal of a methyl group of the allosamizoline moiety of allosamidin decreases the inhibitory effect on family 18 chitinases from three different species (a bacterium, Serratia marcescens, a crustacean, Artemia salina, and an insect cell line, Chironomus tentans). Loss of a second methyl group weakens enzyme inhibition further. This is in agreement with the highly conserved catalytic centre of these enzymes.

Acetylglucosamine↗

PubMed2000

Apoptosis in cultured midgut cells from heliothis virescens larvae exposed to various conditions.

We exposed midgut cells from primary cultures of Heliothis virescens larvae to cell-free previously used medium, the Vaughn X and HyQ SFtrade mark media used for serum-free culture of insect cell lines which do not support H. virescens midgut cells, and to toxin from Bacillus thuringiensis. A statistically significant increase in the percent of dying cells was counted in cell populations in Vaughn X medium. Use of the TUNEL method to detect apoptosis indicated a low rate (7.2%) of apoptosis in control cultures grown in Heliothis medium, an increase to approximately 20% in previously used and HyQ SFtrade mark media, and to approximately 45% of cells remaining after exposure to and initial destruction by B. thuringiensis toxin. Apoptotic nuclei were predominant (approximately 6%) in mature columnar cells in control cultures. Approximately 1% of goblet, stem, and differentiating cells were apoptotic. However, apoptosis rose to 12% in stem and differentiating cells exposed to used and unsuitable medium. B. thuringiensis exposure to toxin for 2-3 days resulted in visible membrane damage and necrosis, causing the death of 84% of the cells as measured by both the TUNEL and Annexin methods. Some of the columnar cells and stem and differentiating cells that remained also contained apoptotic nuclei. Stem and differentiating cells normally replace dying mature cells in the midgut. Thus, exposure of cultures of H. virescens midgut cells to adverse environments such as unsuitable or poisonous media appeared to induce down-regulation of the cell populations by apoptosis.

Animals↗

PubMed2002

The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell-baculovirus expression system.

The effect of dissolved oxygen concentration on human secreted alkaline phosphatase (SEAP) glycosylation by the insect cell-baculovirus expression system was investigated in a well-controlled bioreactor. Oligomannose-type N-linked glycans (i.e., Man2 to Man6 and Man3F) were present in SEAP produced by Spodoptera frusiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines. The relative amounts of the most highly processed glycans (i.e., Man3F and Man2 in the SEAP from Sf-9 and Tn-5B1-4 cells, respectively) were significantly higher at 50% of air saturation than at either 10% or 190% of air saturation. That is, glycan processing was inhibited at both low and high dissolved oxygen concentrations.

Alkaline Phosphatase↗

PubMed2004

Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration.

We describe a simple protocol for determining the oxygen consumption of cells in static culture. The protocol is based on a noninvasive oxygen-sensing microplate and a simple mathematical model derived from Fick's Law. The applicability of the model is confirmed by showing the correlation of computed oxygen consumption rate (OCR) values to actual cell densities ascertained by direct cell counting and/or MTT for HL60 and U937 cells cultured in suspension. Correlation between computed OCR and these other indications of cell number was quite good, as long as the cultures were not diffusion-limited for oxygen. The impact of the geometric factors of media depth and well size were confirmed to be consistent with the model. Based on this demonstrated correlation, we also developed a simple, completely noninvasive algorithm for ascertaining the per-cell oxygen utilization rate (OUR), which is the ratio of OCR to cell number, and a fundamental cell characteristic. This is accomplished by correlating the known seed densities to extrapolated determinations of OCR at time zero. Such determinations were performed for numerous cell types, in varying well sizes. Resulting OUR values are consistent with literature values acquired by far more painstaking methods, and ranged from <0.01 fmol.min(-1).cell(-1) for bacteria to 0.1-10 fmol.min(-1).cell(-1) for immortalized mammalian and insect cell lines to >10 fmol.min(-1).cell(-1) for primary hepatocytes. This protocol for determining OCR and OUR is extremely simple and broadly applicable and can afford rapid, informative, and noninvasive insight into the state of the culture.

Animals↗

PubMedDate not supplied

Semiautomated quantification of cytotoxic damage induced in cultured insect cells exposed to commercial Bacillus thuringiensis biopesticides.

A convenient in vitro bioassay based on semiautomated quantification of live-cell reduction of tetrazolium dyes--3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-corb oxanilide sodium salt (XTT)--to formazan was developed and used to evaluate cytotoxic effects of two commercial insecticides (BT) derived from Bacillus thuringiensis subsp. kurstaki (Btk). Comparison of two target insect cell lines MG1 (Trichoplusia ni, cabbage looper midgut) and Sf9 (Spodoptera frugiperda, fall army worm oocyte) revealed similar cell-dependent responses in mitochondrial-associated electron transport activity. The 50% inhibition of formazan production (ID50) obtained by exposing these cells to 20 microM 2,4-dinitrophenol or 5 mM sodium azide occurred in the range 10(-7)-10(-6) International Units (IU) of BT cell(-1) 24 h(-1). Damage to cell adhesion and cytoarchitecture, revealed by light and electron microscopic analysis, increased with BT exposure and dose. MTT was superior to XTT as a cytotoxic indicator in kinetic studies related to spores, a major component of BT. Unless blocked by antibiotic (gentamicin), vegetative growth resulting from spore germination was the major cause of toxicity. The ID50 exposure time using vegetative Btk cells was approximately 0.1-0.2 times that required for BT spores, with or without intact parasporal proteins present. This difference in exposure is an indirect measure of the time required for spores to germinate and produce vegetative cells. The assay methodology developed here, if linked with suitable target and non-target animal cell types, should have broad application for conducting standardizable estimates of cytotoxic potential of any microbe-based biotechnology product.

Bacillus thuringiensis↗

PubMed1995

CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4.

Human cytochrome CYP3A4 is the most abundant of all the P450s in human liver and is involved in the metabolism of many environmental toxicants and drugs. Kinetic studies with CYP3A4 have been hampered due to low activity of this enzyme obtained from recombinant gene expression systems or difficulty in reconstituting activity with the native enzyme purified from human liver. To overcome these obstacles, we have expressed high levels of catalytically active CYP3A4 and human NADPH-cytochrome P450 reductase (CYPOR) together in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T.ni), via a single recombinant baculovirus carrying both cDNAs (CYP3A4-OR). Microsomes containing recombinant CYP3A4-OR from these cell lines were up to 50-times more active in testosterone 6 beta-hydroxylase activity than recombinant CYP3A4 expressed alone and supplemented with purified rabbit CYPOR. The spectral P450 content of CYP3A4-OR T.ni microsomes was 107 pmol/mg microsomal protein and the cytochrome c reductase activity was 3904 units/mg. Recombinant CYP3A4-OR was catalytically similar to human liver CYP3A4 based on similarities in the testosterone metabolite profile, time course of metabolite formation, Vmax and Km values (for CYP3A4-OR, Vmax was 8.8 nmol/min/mg microsomal protein [70 nmol/min/nmol CYP3A4] and Km was 33 microM), the extent of inhibition by 100 microM troleandomycin (> 75%) in the presence of 25 microM testosterone, and the degree of P450 activation in the presence of 20 microM 7,8-benzoflavone. The coexpression of recombinant cytochrome b5 with CYP3A4-OR did not result in an additional increase in CYP3A4-OR activity.

Animals↗

PubMed1993

Expression of human placental cytochrome P450 aromatase (CYP19) cDNA in insect cells using a luciferase based baculovirus vector.

The cDNA encoding human placental cytochrome P450 aromatase (CYP19) was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression vector system. The recombinant protein product was characterized by Northern and Western blot analyses as well as by direct measurement of aromatase activity. The expressed enzyme proved to be both catalytically active in the presence of P450 reductase and immunologically reactive with polyclonal antibodies raised against human placental aromatase. The activity of aromatase increased 10% after the addition of 0.1 microgram/ml hemin chloride to the culture medium. However, the level of aromatase protein decreased considerably when the concentration of hemin chloride reached 10 micrograms/ml indicating that hemin chloride has toxic effects on the lepidopteran insect cell line. In conclusion, the baculovirus system is suitable for high level expression of functional human placental CYP19.

Animals↗

PubMed1995

High level expression of the IL-1 receptor related T1 receptor in insect cells.

Baculovirus-dependent expression of membrane-associated and secreted, heavily modified proteins in insect cells often results in low yields. To optimize expression of T1, a heavily glycosylated receptor related to Interleukin-1 receptor type I, deletion mutants comprising either the heavily glycosylated extracellular domain or the isolated transmembrane and cytoplasmic portions of the T1 receptor were expressed from recombinant baculoviruses. As shown here, the use of a Baculovirus-derived leader sequence (GP67) in combination with an insect cell line with increased secretory capacity resulted in yields of several mg per liter (10(9) cells) of culture of both purified secretory T1 glycoprotein and membrane-associated T1 receptor.

Amino Acid Sequence↗