Aedes Albopictus Cells Research Articles

Low pH-induced pore formation by spike proteins of enveloped viruses.

Exposure of Aedes albopictus cells infected with Semliki Forest virus (SFV; Togaviridae) to mildly acidic pH (5.6) results in a dramatic increase in the host cell membrane permeability due to pore formation by the virus spike proteins. Identical results were obtained when the cells were infected with two other viruses, Sindbis virus (SIN, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae). This permeability change could also be observed on isolated virions of SFV, SIN and VSV by measuring the influx of propidium iodide, a nucleic acid-specific fluorescent marker, into the virions. This influx was dependent on the presence of the ectodomains of the viral spikes and could be hampered by zinc ions. Furthermore, haemagglutinin, a membrane protein of influenza A virus (Orthomyxoviridae), expressed in Aedes cells induced a change in membrane permeability identical to that induced by the spike proteins of SFV, SIN and VSV when exposed to low pH. Thus acid-induced membrane permeability changes produced by spike proteins of three different virus families could be demonstrated in infected cells as well as in virions. Therefore, the low pH-induced pore formation by viral spike proteins seems to be more than an event specific for togaviruses and might well be an inherent property of enveloped viruses that use the endocytotic pathway to infect a cell.

The fragmentation of incoming Semliki Forest virus nucleocapsids in mosquito (Aedes albopictus) cells might be coupled to virion uncoating.

The fate of Semliki Forest virus (SFV) nucleocapsid, especially the capsid protein (C-protein), was investigated during the early stages of a productive infection in mosquito Aedes albopictus cells. Infection of the cells resulted in a time dependent accumulation of a C-protein derived fragment. This fragmentation of incoming viral nucleocapsid was prevented by NH4Cl, an agent generally used to elevate the pH in acidic intracellular compartments, suggesting that a low intravesicular pH is required for this process. Density gradient analysis of the postnuclear cell lysate demonstrated that the fragmentation was associated with a cellular compartment showing a density of 1.14 +/- 0.02 g/ml. This cellular compartment was devoid from a lysosomal marker enzyme and represented the timely preceding cellular fraction through which SFV passed before encountering a lysosomal fraction. Furthermore, the intracellular distribution of the viral, 3H-uridine-labeled RNA suggested that the same fraction might represent a key cellular compartment in which the separation of the viral RNA from the viral structural proteins is primed. In conclusion, these data lead to the suggestion that the fragmentation of incoming SFV nucleocapsids in Aedes albopictus cells might be the part of the mechanism leading to the release of viral RNA into the cytosol during early stages of productive infection.

Pathogen-derived resistance to dengue type 2 virus in mosquito cells by expression of the premembrane coding region of the viral genome.

The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 (Aedes albopictus) cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus. Northern (RNA) blot analysis confirmed the expression of sense or antisense DEN-2 prM RNA in infected C6/36 cells. PrM protein was demonstrated in cells infected with dsSIN virus expressing DEN-2 sense RNAs by an immunofluorescence assay. C6/36 cells were infected with each dsSIN virus at a multiplicity of infection (MOI) of 50 and challenged 48 h later with DEN-2 virus at an MOI of 0.1. Whereas C6/36 cells infected with a control of dsSIN virus supported high levels of DEN-2 replication, C6/36 cells infected with the dsSIN virus expressing prM antisense RNA were completely resistant to DEN-2 challenge. Cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge. Cells expressing prM protein demonstrated some breakthrough of DEN-2 virus when challenged at an MOI of 10. However, expressed untranslatable sense prM RNA conferred complete protection to challenge at the high MOI.

Stable expression of insect GABA receptors in insect cell lines promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines

We are interested in establishing stably transformed insect cell lines efficiently expressing the insect γ-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selectable marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chloride ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the genetic transformation of non-Drosophilid insects.

Inducibility of a molecular bioreporter system by heavy metals

We have developed a molecular bioreporter model for detecting an invertebrate response to heavy metals. The bioreporter system, pMt2-luc, utilizes a Drosophila melanogaster metallothionein promoter to regulate luciferase expression in stably transformed mosquito cells. The LucC5 clone, which was isolated from pMt2-luc transformed, hygromycin-resistant C6/36 (Aedes albopictus) cells, demonstrated a 12-fold increase in luciferase-specific activity 48 h after exposure to 13 ppm copper (Cu). In addition to Cu, exposure of LucC5 cells to 19 ppm lead (Pb) or 3 ppm mercury (Hg) for 48 h induced luciferase expression threefold and fourfold, respectively. Exposures of up to 30 ppm arsenic (As), 8 ppm cadmium (Cd), 7 ppm chromium (Cr), or 5 ppm nickle (Ni) had no effect on luciferase induction. LucC5 cells exposed to metal mixtures of 13 ppm Cu and 19 ppm Pb yielded an additive response with a 14-fold increase in luciferase expression. When organic chemicals such as phenol (3 ppm) were mixed with 13 ppm Cu, 19 ppm Pb, or 3 ppm Hg a significant reduction in luciferase activity was noted. Additionally, atomic absorption spectroscopy suggested that two of the metals, Cu and Pb, show marked differences in accumulation within the LucC5 cell line.

The Expression of Chloramphenicol Acetyltransferase in Mosquitoes and Mosquito Cells Using a Packaged Sindbis Replicon System

Sindbis (SIN) replicon virus was used to express chloramphenicol acetyltransferase (CAT) in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes. RNA transcribed in vitro from a SIN replicon plasmid (pSINrep5/CAT) and from SIN virus helper constructs (pDH-EB or pDH(26S)5′SIN) was coelectroporated into BHK-21 cells to generate replicon viruses, designated rep5/CAT/EB and rep5/CAT/26S. C6/36 cells infected with rep5/CAT/EB or rep5/CAT/26S virus at a multiplicity of infection of 3, expressed 3.8 × 106 and 6.0 × 106 CAT trimers per cell, respectively, at 2 days postinfection (pi). Both viruses attained peak titers by Day 2 pi. Adult female A. triseriatus mosquitoes were intrathoracically inoculated with 7 × 104 IFU rep5/CAT/EB or 1 × 105 IFU rep5/CAT/26S virus. Virus titers remained at approximately 105 IEU/ml through Day 2 pi and decreased roughly 1 log by Day 10 pi. CAT enzyme activity was detected 2 days pi (rep5/CAT/EB, 1.49 × 10−4 units CAT/10 μg protein; rep5/CAT/26S, 2.03 × 10−5 units CAT/10 μg protein) and remained near these levels through Day 10 pi. CAT was detected in the head, salivary glands, midgut, and ovaries of inoculated mosquitoes by indirect immunofluorescence or CAT activity assays. These results suggest that packaged replicon viruses can be useful for expression of heterologous genes in mosquito cells and whole mosquitoes.

Chemical modification of glutathione S-transferase from C6/36, an Aedes albopictus cell line

The cytosolic glutathione S-transferase from an Aedes albopictus cell line, C6/36, was sensitive to tetranitromethane, phenylglyoxal or pyridoxal phosphate modification, but was unaffected by N-acetylimidazole or diethyl pyrocarbonate. The extent of inactivation of the enzyme by those sensitive reagents was dependent on reagent concentrations but was biphasic in nature and did not follow pseudo-first-order kinetics. Glutathione, ethacrynic acid, S-(hexyl)glutathione, or S-(2,4-di-nitrophenyl)glutathione gave substantial protection of the enzyme from inactivation by these reagents. The modified enzyme showed varying Michaelis constants for glutathione ( K mGSH) or 1-chloro-2,4-dinitrobenzene ( K mCDNB) and a smaller catalytic constant ( k cat. These results indicate the involvement of tyrosine, arginine and lysine residues in the reaction mechanism of the mosquito C6/36 cell glutathione S-transferase. From the results of chemical modification and previous pH studies [Chang G.-G., Tsai L.-N., Tang S.-S., and Wang T.-C. (1994) Arch. Biochem. Biophys. 310, 134–143, we propose that tyrosine residue functions as a general base, promoting ionization of the thiol group of enzyme-bound glutathione and resulting in formation of a more reactive nucleophile thiolate that facilitates conjugation. The essential arginine and lysine residues may participate in maintenance of the correct active center structure.

Evaluation of a viral thymidine kinase gene for suicide selection in transfected mosquito cells.

An Aedes albopictus cell line previously shown to be deficient in thymidine kinase activity was transfected with a thymidine kinase gene (tk) from Herpes simplex virus. Survival of the transfected lines in a 'TK+ selective medium' indicated that the viral gene was actively expressed at a level sufficient to rescue the TK-deficient phenotype of the parent line. Unlike the parental cells, TK+ transformants (TK6:hsv cells) were sensitive to 5-bromodeoxyuridine, and contained DNA corresponding to the constructs introduced by transfection. This TK selection system will facilitate recovery of other cotransfected, non-selectable mosquito genes in cultured cells. Transformed cells were treated with several antiviral drugs to define conditions for a 'suicide selection' system, in which cells expressing the viral thymidine kinase enzyme (TK) under the control of an inducible promoter would be selectively destroyed, whereas cells expressing the endogenous mosquito enzyme would remain relatively unaffected. The anti-herpetic drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) showed greater cytotoxicity against transformed cells expressing the viral enzyme, and less toxicity to wild-type mosquito cells. This cell culture system provides a model for initial evaluation of suicide selection systems that may ultimately be adapted to the mosquito using sex- or tissue-specific promoters to drive expression of heterologous genes.

Evidence for dna endonuclease activity in nuclear extracts from mosquito cells

We describe a deoxyribonuclease activity from nuclear protein extracts of cultured Aedes albopictus mosquito cells. The nuclease cleaved linear and circular double-stranded DNA, first generating 3′ OH single-stranded nicks followed by second strand cleavage, but had little or no exonucleolytic activity. Detection of this activity was optimal at pH 7.1, in the presence of a divalent cation (Mg 2+, Ca 2+, Mn 2+, Ba 2+). In the presence of Mg 2+, Zn 2+, Hg 2+ and Cu 2+ inhibited activity, sulfhydryl reagents and ATP had no effect. At physiological temperatures (18–35°C), linear double-stranded DNA probes were preferentially cleaved near sites containing 3-6 consecutive deoxyadenine/thymine base pairs. Results from salt dependency and drug inhibition studies, combined with inspection of DNA sequence, suggested that DNA structure is among the parameters that determine preferred cleavage sites.

Studies on the replication of Mayaro virus grown in interferon treated cells.

Mayaro virus grown in interferon treated infected cells has been characterized with regard to its ability to replicate in vertebrate (TC7) and invertebrate (Aedes albopictus) cells. Virus purified from interferon treated TC7 cells adsorbs and penetrates to the same extent as the control virus. During infection, these virus particles caused inhibition of host protein synthesis and synthesized the same spectrum of viral proteins as normal virus. This population however, was apparently more sensitive to interferon treatment. Electron microscopy of TC7 cells showed the presence of numerous aberrant virus particles budding from the plasma membrane.

Structure, restriction map and infectivity of the genomic and replicative forms of AaPV DNA.

We have characterized the genomic and replicative form (RF) DNA of the Aedes albopictus Parvovirus (AaPV), a virus isolated from a chronically infected C6/36 clone of Aedes albopictus cell line [22]. The genome of AaPV virions is a single-stranded linear DNA molecule approximately 4.2 kb in length, essentially (about 90%) encapsidated as minus strand. A restriction map of the RF DNA isolated from infected C6/36 cells was established. Among the 23 restriction enzymes tested, 14 cleaved the AaPV RF DNA and 30 restriction sites were mapped and oriented with respect to the viral genomic DNA. Both viral and RF DNAs were found infectious when transfected to virus-free C6/36 cells. The asymmetrical encapsidation of the viral genome is a property common to most vertebrate autonomous parvoviruses but rather unusual among densoviruses. Both by its small size, the asymmetrical mode of encapsidation and the restriction map, the AaPV genome resembles that of the Aedes Densonucleosis virus [1].

A 55-kDa Protein Induced in Aedes Albopictus (Mosquito) Cells by Antiviral Protein

We have previously shown that the antiviral protein (AVP) produced by Sindbis virus-infected Aedes albopictus (mosquito) cells (Riedel and Brown, J. Virol. 29, 51-60, 1979) blocks Sindbis viral RNA synthesis and stimulates its own production when applied to uninfected A. albopictus cells (Luo and Brown, Virology 194, 44-49, 1993). From a virus-sensitive (wild-type) mosquito cell line (U4.4) we produced a virus-resistant cell line (L4.4) by exposing U4.4 cells to the AVP. L4.4 cells constitutively produce AVP and fail to replicate viral RNA after infection with virus or transfection with purified viral RNA. In this study we have compared cellular proteins produced as the sensitive cells are temporally converted to the resistant phenotype following exposure to the AVP. A 55-kDa membrane protein associated with lysosomes is found in L4.4 and not in U4.4 cells. This protein is induced during the first 48 hr following treatment of U4.4 cells with the AVP. The correlation of the appearance of the 55-kDa protein with the onset of virus resistance implies that this AVP-induced protein may be responsible for the inhibition of viral RNA replication. Although virus RNA synthesis is blocked in the L4.4 cell line, we have found that the synthesis of nonstructural proteins takes place.

Secretion of an Inducible Cecropin-Like Activity by Cultured Mosquito Cells

Characterization of activities that provide potential targets for genetic manipulation of pathogen development, maintenance, and transmission in transgenic insects has applications to the eventual control of malaria and other arthropod-transmitted diseases. We have identified inducible activities from cultured mosquito (Aedes albopictus) cells, including one that shares the antimicrobial properties of cecropins from other insects. The cecropin-like activity can be induced by treatment with heat-killed Escherichia coli, is secreted into the cell culture medium, and can be detected after electrophoresis of acid-precipitable proteins on polyacrylamide gels at pH 4.3. Cecropin lacks the amino acids methionine and cysteine. Other proteins secreted in response to bacterial induction measure 111, 66, 53, and 32 kD; these proteins incorporate sulfur-containing amino acids and were detected on denaturing polyacrylamide gels. The synthesis of antimicrobial proteins by mosquito cells in culture will contribute to an understanding of the diversity of molecules that participate in insect immunity and to the use of continuous cell lines and their inducible products to explore and manipulate regulation of physiological processes relevant to vector biology.

Intracellular immunization of mosquito cells to LaCrosse virus using a recombinant Sindbis virus vector.

A cDNA of the small RNA genome segment of La Crosse (LAC) virus was inserted, in an antisense orientation, into a double subgenomic Sindbis (dsSIN) virus expression vector generating pTE/3'2J/ANTI-S (15,000bp). In vitro transcription of the pTE/3'2J/ANTI-S template generated genomic RNA that was electrotransfected into BHK-21 cells to produce virus. Northern blot analysis of RNA isolated from infected Aedes albopictus (C6/36) cells showed that the TE/3'2J/ANTI-S virus produced a subgenomic mRNA of the appropriate size, indicating transcription of the LAC cDNA segment. C6/36 cells were infected with either TE/3'2J/ANTI-S, TE/3'2J (a dsSIN virus with no LAC insert), or wild type Sindbis (SIN, strain AR339) viruses and subsequently challenged with LAC virus. LAC virus titers were determined using a capture antibody ELISA. Mosquito cells infected with TE/3'2J/ANTI-S virus yielded at least 4 log10 TCID50/ml less LAC virus than cells infected with either TE/3'2J or AR339 SIN viruses. The use of the infectious SIN virus expression vectors provides a novel approach for high level cytoplasmic expression of genes or sequences of interest in arthropod cells, and for evaluating strategies for intracellular immunization against arboviruses.

Expression of an antisense dihydrofolate reductase transcript in transfected mosquito cells: effects on growth and plating efficiency.

Novel approaches to control of vector-borne disease include potential use of transgenic insects, in which molecular mechanisms will be induced to prevent transmission of pathogenic organisms. The infrastructure essential to this technology includes the cloning of essential genes from vector insects, and the development of efficient transformation strategies. In this study, we use a continuous mosquito (Aedes albopictus) cell line and a cloned mosquito dihydrofolate reductase gene to demonstrate a transgenic approach that may be used to select for the presence or absence of particular gene functions in transfected cells. Plasmids containing the dihydrofolate reductase gene in sense and antisense orientation, under the regulation of a temperature-inducible promoter, were expressed in stably transfected mosquito cells. At the normal growth temperature of 28 degrees C, or after mild heat induction at 34 degrees C, expression of the dihydrofolate reductase construct in sense orientation had little effect on cell growth. In contrast, recovery of clones transfected with the antisense construct was reduced, and induction of antisense transcripts at 34 degrees C further compromised cell growth and viability. Clones transfected with the sense construct retained significantly higher copy numbers of foreign DNA than did cells transfected with the antisense construct. These studies provide a basis for use of sense and antisense dihydrofolate reductase constructs to recover transfected mosquito cells with specific desired phenotypes, based on the relative expression of cloned genes of interest.

Purification and Kinetic Mechanism of the Glutathione S-Transferase from C6/36, an Aedes albopictus Cell Line

Glutathione S-transferase from an Aedes albopictus cell line, C6/36, was purified to apparent homogeneity by a single glutathione-Sepharose 4B affinity chromatography, with an overall yield of 66% and 226-fold purification. The enzyme is presumably a homodimer with subunit Mr 23,000, which is similar to the enzyme isolated from other sources. Under native conditions, the enzyme exhibited multiple forms upon isoelectric focusing or polyacrylamide gel electrophoresis, and all these forms retained enzymatic activity. The pI value of the purified enzyme was distributed between ∼4.7 and 5.4 with major form at 4.9. The purified enzyme lost 63% activity at −20°C within 1 week. Stability of the purified enzyme was greatly improved by the addition 10 mg/ml of bovine serum albumin, under which only 7% activity was lost after 1 week at −20°C. Activation energy for the enzyme-catalyzed conjugation of glutathione with 1-chloro-2,4-dinitrobenzene was found to be 49.1 kJ/mol. The enzyme could utilize 1-chloro-2,4-dinitrobenzene and ethacrynic acid as substrate, but not bromosulfophthalein, cumene hydroperoxide, or trans-4-phenyl-3-buten-2-one. The initial-velocity and product-inhibition studies indicated that the enzyme reaction conformed to a steady-state random Bi-Bi kinetic mechanism, which was similar to the glutathione S-transferase from other sources. The kinetic data also indicated a synergistic effect between the binding of glutathione G-site and that of organic electrophilic substrate in the H-site of the enzyme active center. The biological significance of the conjugation reaction is discussed.

The expression of chloramphenicol acetyltransferase in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes using a double subgenomic recombinant sindbis virus

Genomic RNA was transcribed in vitro from the double subgenomic recombinant Sindbis (SIN) virus expression vector, pTE/3′2J/CAT, and transfected into BHK-21 cells to generate recombinant virus stocks. TE/3′2J/CAT virus was used to infect C6/36 ( Aedes albopictus) cells and adult female Aedes triseriatus. When C6/36 cells were infected with TE/3′2J/CAT virus at a multiplicity of infection (MOI) of greater than 20, 100% of the cells expressed CAT. The number of CAT polypeptides expressed per cell at 24 h post infection (pi) was 8.3 × 10 5. Approximately 4.0 log 10 TCID 50 of the TE/3′2J/CAT virus was intrathoracically inoculated into adult female mosquitoes. Titers greater than 6.0 log 10 TCID 50/ml were detected within 4 days pi and declined to less than 4.0 log 10 TCID 50/ml 20 days following inoculation. CAT activity was detected within 2 days (8 × 10 −5 units of CAT/mosquito or 1.4 × 10 10 CAT polypeptides), peaked at day 6 (4 × 10 −3 units of CAT/mosquito or 7.2 × 10 11 CAT polypeptides), and remained at peak levels to day 20. Immunofluorescence and CAT activity assays were used to localize CAT expression in infected mosquitoes and demonstrated that CAT was present in neural, midgut, ovarian, and salivary gland tissues. Alphavirus-based expression vectors should be useful for expressing heterologous genes in mosquito cells as well as adult mosquitoes.

Mutations in the nsP1 Coding Sequence of Sindbis Virus Which Restrict Viral Replication in Secondary Cultures of Chick Embryo Fibroblasts Prepared from Aged Primary Cultures

SVMPA, a mutant of Sindbis virus (SV), which is able to replicate in Aedes albopictus cells treated with mycophenolic acid (MPA) or ribavirin, also has a host range phenotype. This phenotype is most clearly demonstrated by means of efficiency of plaquing (EOP) assays on secondary chick embryo fibroblasts (CEF) prepared from aged primary CEF. For example, in one experiment in which standard SV (SVSTD) had a relative EOP (EOP on primary CEF ÷ EOP on secondary CEF) of 1.6 the corresponding value for SVMPA was 2340. The host restriction of SVMPA was also seen with similarly prepared secondary cultures of duck embryo fibroblasts, but not with established lines of quail cells. The finding that the accumulation of viral RNA was much lower in SVMPA-infected secondary CEF than in SVSTD-infected CEF indicated that the replication of SVMPA in these cultures was blocked at an early step. Revertants of SVMPA were isolated which were no longer host-restricted but had retained their resistance to MPA. Of the three mutations [nucleotide (nt) 120, 127, and 963] in the nsP1 coding sequence of SVMPA, which lead to amino acid changes, these revertants had retained the nt 127 and nt 963 mutations but had lost the nt 120 mutation. This, along with earlier findings, indicated that only the nt 127 and nt 963 mutations are required for resistance to MPA. This result also associated the nt 120 mutation with the host restriction phenotype. In other experiments derivatives of pToto1101 (a plasmid from which infectious Sindbis virus RNA can be transcribed) were constructed by site-directed mutagenesis and used to test the effect of specific mutations on the viral phenotype. Although we were unable to obtain viable virus with the nt 120 mutation alone, virus with the nt 120 and nt 127 mutations was viable and host-restricted. We suggest that the nt 120 mutation by itself is lethal and that the nt 127 mutation suppresses the lethal effect of the nt 120 mutation. The SVMPA mutation at nt 120, which is associated with the host range phenotype, changes Gln21 of nsP1 to Lys. When a more conservative change was engineered, i.e., to Asn, the virus was not host-restricted. Although the reason for the restriction of SVMPA replication in secondary CEF is not known, some possible explanations are discussed.

Growth and maintenance of aedes albopictus cell line , clone c6/36 , in different media

The sensitivity of Aedes albopicts cell line, clone C6/36, to arbovirus isolation and growth has been being shown by several authors. The present work has verified which, among several media under the same conditions, would be the most efficient one for C6/36 cultivation and viral isolation. The L-15 medium has proved to be the best among others (MEM, DMEM and 199) with the quickest viral isolation (DEN-1). Also, in this medium, the samples could be observed until the 14th day, without significative cellular death. The results reccommend L-15 medium as the most efficient and economic one for the purposes.

Reversal of the antiviral activity of ribavirin against Sindbis virus in Ae. albopictus mosquito cells

Earlier work in our laboratory has shown that the replication of Sindbis virus in Aedes albopictus mosquito cells is inhibited by ribavirin (Rbv) and mycophenolic acid (MPA) (Sarver and Stollar (1978) Virology 91, 267–282; Malinoski and Stollar (1980) Virology 102, 473–476). We report here that the antiviral effect of Rbv and MPA can be reversed by depriving infected cells of methionine or isoleucine, or by treating them with fluorodeoxyuridine (FUdR) or cycloleucine. We suggest that, as was the case when the antiviral activity of Rbv was reversed by actinomycin D (Malinoski and Stollar (1981a) Virology 110, 281–291), these effects may be mediated by changes in the GTP pools of treated cells.