Aedes Albopictus Cell Line Research Articles

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.

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].

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.

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.

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.

A Parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae

We have isolated and partially characterized from an apparently healthy C6/36 subclone of Aedes albopictus cell line a small icosahedral non-enveloped DNA virus, designated AaPV. This virus proved to be highly pathogenic for Aedes aegypti neonate larvae. Viral infection persisted for over 4 years in the cell culture without any cytopathic effect. Attempts to infect suckling mice, Drosophila melanogaster adults and Spodoptera littoralis larvae with AaPV were unsuccessful. Similarly, the AaPV failed to replicate in vertebrate and Drosophila cell lines. Virions, about 22 nm in diameter, had a buoyant density of 1.43 g/cm 3 and contained three capsid polypeptides with molecular weights of 53, 41 and 40 kDa. A preliminary study of the viral genome indicated the presence of single-stranded DNA. By its biophysical and biochemical properties, this virus appears to be related to the genus Densovirus within the family Parvoviridae, but Jacks serological relationships with the other members of this genus.

Factors affecting zero background frequency of sister-chromatid exchange in mosquito cells

A background level of sister-chromatid exchange (SCE) is found in mammalian cells. However, in the somatic cells of Drosophila melanogaster, there was no spontaneous SCE if a minimum concentration of bromodeoxyuridine (BrdU) was used. In this communication we report that in a mosquito (Aedes albopictus) cell line (C6/36), some cells also have no background SCE (0-SCE). Subclones of high (44%) or low (12%) frequency of 0-SCE cells were obtained, but none of them contained 100% 0-SCE cells. Increasing frequency of SCE/cell concomitant with decreasing frequency of 0-SCE cells was observed by raising the BrdU concentration in the culture medium during the first cell cycle, culturing cells at lower density and depleting reduced glutathione (GSH) with buthionine sulfoximine. Since these mosquito cells contain much higher level of GSH than Chinese hamster ovary (CHO) cells, and feeding cells with BrdU increased GSH in mosquito cells but decreased GSH in CHO cells, we speculate that GSH may play a role in the low or non-existent background SCE and in the high resistance to many DNA damaging agents of mosquito cells.

Unusual morphology of a virus which produces carbon dioxide sensitivity in mosquitoes

A virus, named Matsu, presumed to be the etiologic agent of hereditary sensitivity to carbon dioxide in Culex quinquefasciatus mosquitoes, was adapted to growth in the C6/36 line of Aedes albopictus cells. Though it was expected that the mosquito virus would be a rhabdovirus like sigma, the etiologic agent of hereditary carbon dioxide sensitivity in Drosophila melanogaster flies, that was not the case. The virion of Matsu was found to be unlike any previously described virus. It was pleomorphic, enveloped, from 200 to 550 nm in maximum diameter, and contained from three to several dozen virus-like polyhedral structures approximately 30 nm in diameter.

A single amino acid change in the E2 glycoprotein of Venezuelan equine encephalitis virus affects replication and dissemination in Aedes aegypti mosquitoes

Four monoclonal antibody-resistant variants (MARVs) of Venezuelan equine encephalitis (VEE) virus were used to study mosquito-virus interactions. In vitro experiments using an Aedes albopictus cell line, C6/36, demonstrated that an amino acid change in the glycoprotein E2h epitope (MARV 1A3B-7) decreased virus growth when compared with the wild-type, Trinidad donkey virus, and its vaccine derivative, TC-83. The MARVs replicated as efficiently as the parent virus when inoculated into Aedes aegypti mosquitoes, but MARV 1A3B-7 was restricted in its ability to infect and disseminate from the midgut following oral infection. These results demonstrate that a single amino acid change in the E2 glycoprotein can affect the ability of VEE virus to replicate and disseminate in Ae. aegypti mosquitoes.

Effect of actinomycin D and cycloheximide on replication of Sindbis virus in Aedes albopictus (mosquito) cells.

Production of Sindbis virus in the presence of transcription and translation inhibitors was examined in three Aedes albopictus cell lines. Addition of cycloheximide to heat-resistant Sindbis virus (SVHR)-infected mosquito cells arrested viral RNA synthesis completely, in contrast to the effects of this drug on virus-infected vertebrate cells. Production of mature virus by both SVHR (a variant commonly used as a wild-type virus) and SBamr (a mutant which is resistant to the effects of 18 h of pretreatment of vertebrate cells with actinomycin D) in mosquito u4.4, C6-36, and C7-10 cells was inhibited by 2 h of pretreatment with actinomycin D. Pretreatment with this drug for 2 h slightly enhances virus production in vertebrate cells. Treatment of mosquito cells with actinomycin D resulted in shutoff of SVHR RNA synthesis. The mutant SBamr was able to overcome the effects of actinomycin D on viral RNA synthesis and produced both 26S and 49S RNAs, even though no viral structural proteins or mature particles were produced in the presence of the drug. This result suggests that, in the presence of actinomycin, the nonstructural genes of SBamr are translated sufficiently to allow for RNA synthesis but that 26S RNA may not be translated to an extent that allows significant virus production. These data demonstrate that host components are involved in at least two distinct steps in the production of Sindbis virus in mosquito cells: (i) production of viral RNA and (ii) synthesis of viral structural polypeptides.

Suppression of RNA synthesis by a specific antiviral activity in Sindbis virus-infected Aedes albopictus cells.

An antiviral protein is released by mosquito cells persistently infected with Sindbis virus. Differences in both sensitivity to and production of this virus-specific activity were apparent in three independently produced Aedes albopictus cell lines. This activity inhibits total viral RNA synthesis in a time-dependent manner. The antiviral effect is maximally realized when cells are treated with the activity 48 h before infections. These data suggest that the antiviral activity induces an antiviral state in treated cells which prevents the formation or efficient function of viral RNA-synthesizing complexes.

Prolonged infection of human synovial cells with Ross River virus.

Primary cultures of human synovial cells shed infectious virus for 14 to 35 days following infection with isolates of Ross River virus which had been passaged in the C6/36 line of Aedes albopictus mosquito cells. No frank cytopathic effect was seen in infected synovial cells and they continued to replicate for the duration of the experiments.

Expression of recombinant vaccinia virus-derived alphavirus proteins in mosquito cells.

A recombinant vaccinia virus strain which contains and expresses a 26S cDNA insert encoding Sindbis virus structural proteins (VV:3S) was used to infect a continuous line of Aedes albopictus mosquito cells. There were not visible cytopathic effects due to the virus infection and the cells continued to grow normally. However, examination of the proteins present in the cytoplasm of the infected cells with Sindbis virus-specific antisera revealed that Sindbis virus proteins were being synthesized and processed. These results are discussed with respect to vaccinia virus as a non-lethal expression vector to deliver and express eukaryotic genetic information in insect cell systems and using this system (VV:3S) to dissect various facets of togavirus-insect cell interactions.

Replication pattern of double minutes derived from an insect cell line.

The DNA replication pattern of double minutes derived from an established cell line of Aedes albopictus is described. Although the vast majority of double minutes replicate semiconservatively once during the S phase, some double minutes appear to exhibit different pattern(s). Two theories are suggested as possible explanations of our findings.

A simplified new method for the identification of arboviruses: Virus detection using enzyme immunoassay on cultured cells

A simple new method is reported for the identification of arbovirus isolates and the titration of arboviruses. Horseradish peroxidase conjugated Staphylococcus aureus protein A was used for the indirect staining of virus antigens grown in an Aedes albopictus cell line. C6/36 cells. Thirty-five isolates from Culex tritaeniorhynchus mosquitoes were examined by this method and 20 of these were identified as Japanese encephalitis virus (JEV). These results were confirmed by immunofluorescent assay and plaque neutralization test. Comparative titration of JEV, Murray Valley encephalitis (MVE) and Kunjin viruses showed that this method was as sensitive as the chick embryo plaque assay. Specific enzymatic reactions on these virus-infected cells began to appear before day 3 and reached the end-point on day 5 post-infection of C6/36 cells, whereas the cytopathic effect (CPE) appeared about 2 days later than the positive enzymatic reactions. Fixation with 10% formalin for 0.5–8 h did not damage the positive reaction in infected cells and did not increase the background colour of uninfected cells.

Properties of Sindbis virus variants from infected Culex tarsalis mosquitoes.

Plaque-purified Sindbis virus was passed three times in Culex tarsalis mosquitoes and progeny viruses were isolated by plaque purification on a cloned line of Aedes albopictus cells. Nine of ten clones examined differed from wild-type (wt) virus with respect to their plaque morphology characteristics in chick embryo fibroblast (CEF) and/or A. albopictus cells. Seven clones were temperature-sensitive and failed to replicate or synthesize viral RNA in CEF cells at 41 degrees C. At 35 degrees C in CEF cells the majority of isolates synthesized less viral RNA than wt virus. In contrast, all cloned isolates synthesized viral RNA more rapidly than wild-type virus in A. albopictus cells.

Methotrexate resistance in cultured mosquito cells

Several mosquito ( Aedes albopictus) cell lines resistant to methotrexate, an inhibitor of the enzyme dihydrofolate reductase, have been characterized. On the basis of growth in the presence of methotrexate, the initial variants, Mtx-101, Mtx-102, Mtx-111, and Mtx-501, were 6.5-fold more resistant to methotrexate than the parental cells. From these cell lines, clones with increased resistance to methotrexate were selected. The second-level variants Mtx-5013 and Mtx-5011, were, respectively, 70- and 200-fold more resistant to methotrexate than wild type cells, and they contained 14- to 20-fold more dihydrofolate reductase activity. In extracts from Mtx-5013 and Mtx-5011 cells, the apparent affinity of dihydrofolate reductase for methotrexate was reduced about 20-fold with respect to that of wild type cells. Resistant cell lines became more sensitive to methotrexate during long-term passage in non-selective medium. This is the first description of methotrexate-resistant variants derived from cultured insect cells.

Isoprene synthesis in isolated embryonic Drosophila cells. I. Sterol-deficient eukaryotic cells.

Since insects are cholesterol auxotrophs, we analyzed the apparent paradox presented by an established cell line (Kc cells) from Drosophila embryos which grew in media which contained less than 0.05 micrograms/ml of sterols. Fresh Drosophila embryos contained 3.7 micrograms of 3 beta-hydroxysterols/mg of protein; however, Kc cells had maximally 0.50 micrograms of 3 beta-hydroxysterols/mg of protein. Kc cells, grown in media which contained cholesterol, showed the presence of sterol in their plasma and intracellular membranes. Kc cells did not synthesize sterols or any apparent replacement lipophilic molecule. However, two major compounds which comigrated with ubiquinone and dolichol were synthesized from radioactive mevalonate and acetate. Cholesterol incorporation into Kc cell membranes did not significantly alter total phospholipid head or acyl group composition. Similar observations were obtained with Schneider's Drosophila cell line I and a mosquito (Aedes albopictus) cell line. Our results (a) challenged current concepts that sterols or related replacement isopentenoid molecules were required for eukaryotic membrane structure, (b) demonstrated that marked alterations in eukaryotic membrane sterol composition was insufficient to change total phospholipid head and/or acyl group composition, and (c) set the stage for the use of a eukaryotic cell to examine the regulation of 3-hydroxy,3-methylglutaryl coenzyme A reductase activity independent of a requirement for sterol synthesis.

Isolation and characterization of puromycin-resistant clones from cultured mosquito cells.

We have isolated from an established Aedes albopictus (mosquito) cell line clones which are resistant to the antibiotic puromycin. On the basis of growth and plating efficiency, clones Pur-8026 and Pur-8612 were five- and seven-fold more resistant, respectively, to puromycin than wild-type cells. In vitro protein synthesis was resistant to puromycin only in extracts prepared from Pur-8612 cells. Measurements of puromycin transport, cross-resistance to colchicine, and sensitivity to Tween-80 indicating that resistance in Pur-8026 cells was due to membrane alteration(s) affecting permeability to puromycin. This is the first description of puromycin resistant in insect cells and also the first report of puromycin resistance in an animal cell variant associated with an alteration at the level of protein synthesis.

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.