Aedes Albopictus Cells Research Articles

Investigation of the role of glycans for the biological activity of Semliki Forest virus grown in Aedes albopictus cells using inhibitors of asparagine-linked oligosaccharides trimming.

The effects of N-linked-oligosaccharide-processing inhibitors on the formation of Semliki Forest virus (SFV) in C6/36 Aedes albopictus cells were investigated. The glycosidase inhibitors deoxynojirimycin, deoxymannojirimycin and swainsonine prevented the formation of Endo-H resistant structures, but had little effect on virus formation and on the biological activities of the virus. Tunicamycin greatly inhibited virus formation, but had little effect on cell-cell fusion from within and the cleavage of p 62. These results indicate that correct glycosylation is not a prerequisite for biological activities of SFV, whereas glycosylation per se is needed for virus production.

SV LM21, a mutant of sindbis cirus able to grow in Aedes albopictus cells in the absence of methionine, shows increased sensitivity to S-adenosylhomocysteine hydrolase inhibitors such as neplanocin A

Inhibition of S-adenosylhomocysteine (AdoHcy) hydrolase by compounds such as Neplanocin A (NPA) leads to the build-up of AdoHcy and the inhibition of methyltransferase enzymes. Whether assayed by efficiency of plaquing or virus yield, SV LM21, a mutant of Sindbis virus resistant to methionine deprivation, was more sensitive to NPA than was the standard virus (SV std) from which it was derived. For example, whereas 10 μg NPA/ml depressed the yield of SV LM21 by more than 30-fold, the yield of SV std was not significantly affected. Similar differences in sensitivities were shown to three other compounds which inhibit AdoHcy hydrolase. These results support the idea that SV LM21 codes for an altered RNA methyltransferase.

Exposure of Semliki Forest virus-infected baby hamster kidney cells to low pH leads to a proton influx and a rapid depletion of intracellular ATP which in turn prevents cell-cell fusion. Brief report.

The conditions needed to trigger Semliki Forest virus (SFV) induced cell-cell fusion from within (FFWI) of baby hamster kidney (BHK) cells differs from some other cells (e.g., Aedes albopictus cells). In contrast to such cells it is mandatory for BHK cells to be brought back to a neutral pH after a short exposure to mildly acidic pH for fusion to occur. This phenomenon can be explained by a sudden drop of the intracellular pH observed after clamping the extracellular pH below 6.2--the pH required to trigger fusion--and by the thereby implied increased expenditure of ATP. Since the fusion from within of SFV-infected cells is energy dependent [Kempf et al. (1987) Arch Virol 95: 111-122] the fusion process is blocked due to the ATP depletion.

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.

Absence of antiviral activity of the broad-spectrum picornavirus inhibitor disoxaril (WIN 51711) against hepatitis A virus : Anders Widell, Laboratory of Clinical Virology, University of Lund, Malmö General Hospital, Malmö, Sweden

The replication of Sindbis virus (SVSTD) in cultured Aedes albopictus mosquito cells is sensitive to methionine deprivation. We have suggested from earlier work that this sensitivity is primarily because of a decreased pool of S-adenosyl methionine (ado met) and the resultant failure to methylate the 5′ cap of the viral mRNAs. SVLM21, a strain of Sindbis virus derived in our laboratory from SVSTD by serial passage on mosquito cells maintained after infection in low concentrations of methionine, is resistant to methionine starvation. It was proposed that this adaptation to low methionine, and to the resultant low intracellular levels of ado met, reflected the accumulation of mutations which led to the generation of a viral RNA cap methyltransferase with an increased affinity for ado met. We report here kinetic data which distinguished the enzymes coded for by SVSTD and SVLM21. Using guanylylimidodiphosphate (GIDP) as the methyl acceptor, radioactively labeled ado met as the methyl donor, and lysates from infected BHK cells as the enzyme source, we calculated from our results that SVLM21, generated a methyltransferase with a Km for ado met 10-fold lower than that generated by either SVSTD or the related alphavirus, Semliki Forest virus. In addition, we found that BHK cells infected with SVLM21, generated higher levels of methyltransferase activity than did cells infected with SVSTD and that the SVSTD and SVLM21, enzymes differed with respect to their relative activities at elevated temperatures. We conclude from these results that the SVLM21, phenotype is associated with an altered methyltransferase and suggest that this is the basis of the resistance of SVLM21, to methionine deprivation.

Growth of bluetongue and epizootic hemorrhagic disease of deer viruses in poikilothermic cell systems

Continuous cell lines from the ticks Dermacentor variabilis, D. parumapertus, D. nitens, Rhipicephalus sanguineus and R. appendiculatus, the mosquitoes Aedes albopictus and Culex quinquefasciatus and the African toad Xenopus laevis were tested for their ability to replicate bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses, and for their sensitivity as potential isolation systems. BT serotype 17 grew to peak titers of 10 4.5−10 7.5 TCID 50 ml −1 in all except one of the tick cell lines, EHD 2 virus attained titers similar to that of BT 17 in the mosquito and toad cells, but failed to replicate in tick cells. Only Aedes albopictus and Xenopus laevis cells were as sensitive to infection with low-passage BT 11 and EHD 2 viruses as control cultures of Vero and BHK cells. At 27°C, persistent infection of Xenopus laevis cells occurred, producing low yields of BT 17 and EHD 2. When shifted to 32°C, these cultures expressed virus in exponential increments. No cytopathic effect (CPE) was seen in any of the tick-virus systems, but infected mosquito and toad cells detached from the monolayer within 3–6 days after inoculation with either virus. In the toad cells, this CPE was presaged by the development of plaques within 48 h after infection. Potential applications of poikilotherm systems in orbivirus research are discussed.

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.

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture.

An insecticidal protein gene from Bacillus thuringiensis var. aizawai was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteolytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resistant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CF1 cells but not dipteran (Aedes albopictus) cells. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawai toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran cell membranes, which may be the receptor for this toxin.

Cultivation in mosquito cells of sigma virus, the aetiologic agent of hereditary CO 2 sensitivity in Drosophila melanogaster

Summary Sigma virus was adapted to growth in the C6/36 clone of Aedes albopictus cells after 3 serial passages in intact Toxorhynchites amboinensis mosquitoes. The virus replicated when the cells were held at 20 and 25°C, but not when they were held at 36°C. Virus was liberated into the supernatant fluid of cultures — which sometimes contained as much as 10 8.0 infectious units per ml. After a high-titred antiserum had been prepared using purified virus grown in cell cultures, it was possible to isolate sigma virus directly in the cell cultures from triturates of infected Drosophila melanogaster flies by using an indirect fluorescent antibody technique as an indicator system. Cell cultures could also be employed for titration of the virus using the same indicator system. This method of viral assay was as sensitive as the inoculation of intact D. melanogaster — at least for cell-culture-adapted virus.

A novel method for the detection of early events in cell-cell fusion of Semliki Forest virus infected cells growing in monolayer cultures.

SummarySemliki Forest virus infected Aedes albopictus cells were used to investigate virus induced cell-cell fusion. It was shown by a novel method that cell-cell fusion was completed within approximately 5 minutes after triggering the fusion event by low pH. This method consists of fixing fusing cells with glutaraldehyde and microinjecting the highly fluroescent and rapidly diffusing dye Lucifer yellow. In contrast, polykaryon formation, the usually used criterion to measure cell-cell fusion occurred only within 30 minutes. Furthermore, it was shown that potassium cyanide, a potent inhibitor of polykaryon formation in the described system, inhibits an early step of membrane-membrane fusion of neighbouring cells.

Susceptibility of Aedes albopictus C6/36 cells to viral infection.

The susceptibility of the C6/36 clone of Aedes albopictus monolayer cell cultures was determined with 46 prototype viruses passed through three subcultures. Viral growth was confirmed by titration of the passage material in other susceptible host systems. Nineteen viruses demonstrated good growth in C6/36 cells: coxsackievirus group A type 10 and group B types 2, 3, 4, and 5; enterovirus 69; mumps virus; poliovirus types 1 to 3; reovirus types 1 to 3; vaccinia virus; dengue virus type 2; eastern equine encephalomyelitis virus; La Crosse virus; Rocio virus; and St. Louis encephalitis virus. Ten viruses did not adapt to growth in the C6/36 cultures. Seventeen other virus strains displayed only limited growth which was primarily restricted to the initial C6/36 passage or was detected by hemagglutinin reactions without observable cell degeneration. Of the 46 viruses, 33 (72%) were capable of initiating infection with a demonstrable cytopathic effect in the initial C6/36 passage. Hemagglutination or complement fixation titers or both were obtained with dengue virus type 2, eastern equine encephalomyelitis virus, La Crosse virus, mumps virus, reovirus types 1 to 3, and Rocio, St. Louis encephalitis, and vaccinia viruses.

Ribosome metabolism during the vitellogenic cycle of the mosquito, Aedes aegypti

Ribosome accumulation and synthesis in the fat body of the mosquito Aedes aegypti increased by approx. 4-fold during 18 h after blood meal, consistent with the pattern of total RNA accumulation during the synthetic phase of the vitellogenic cycle. By 24–30 h after the blood meal, the accumulated ribosomes began to be degraded, and the total RNA content in the fat body eventually returned to previtellogenic levels. A method has been developed for isolation of ribosomal subunits from fat body, and the ribosomal proteins have been shown to have properties similar to those from cultured Aedes albopictus cells by two-dimensional polyacrylamide gel electrophoresis. Proteins S1, S10, and A from the small ribosomal subunit are phosphorylated when a fat body is incubated in Aedes saline in the presence of [ 32P]orthophosphate.

The surface charge of L-A9 cells and Aedes albopictus cells infected with Marituba (Bunyaviridae) virus

The surface charge of Marituba virus infected L-A9 cells and Aedes albopictus cells was estimated by direct measurement of their electrophoretic mobilities. Uninfected L-A9 cells and A. albopictus cells have mean electrophoretic mobilities of −1.083 μm/s × cm/V and −1.019 μm/s × cm/V, respectively. In Marituba virus infected L-A9 cells a progressive decline in the electrophoretic mobility was observed. In contrast, in Marituba virus infected A. albopictus cells the electrophoretic mobility of the cell surface,was unaltered.

N‐terminal amino acid sequence analysis of dihydrofolate reductase from methotrexate‐resistant mosquito cells

AbstractMethotrexate‐resistant mosquito (Aedes albopictus) cell mutants were used as starting material for the purification of dihydrofolate reductase. Initial fractionation of a crude cell lysate by ammonium sulfate precipitation showed an enrichment of enzyme activity in the 60‐100% pellet. Purification of this ammonium sulfate fraction on a Sephadex G‐75 column prior to affinity chromatography was required for maximal yield of purified protein. A protein with dihydrofolate reductase activity and an Mr of 24,000 was eluted from a methotrexate‐agarose column with 0.5 M Tris‐HCl, pH 8.5, containing 2 mM folic acid. A 21‐fold increase in specific activity relative to that in the crude cell lysate was obtained, and amino acid sequence analysis confirmed that dihydrofolate reductase from the affinity column migrated as an electrophoretically pure band on SDS polyacrylamide gels. The initial 20 amino acid residues of the mosquito dihydrofolate reductase were identified by microsequencing and compared to those of murine, human, and bacterial dihydrofolate reductase molecules.

Sindbis virus mutants resistant to mycophenolic acid and ribavirin

Previous work from this laboratory has demonstrated a correlation between the inhibition by ribavirin (Rbv), mycophenolic acid (MPA), or 2-amino thiadiazole (TDA) of Sindbis virus replication in Aedes albopictus mosquito cells and a reduction in cellular GTP levels. This reduction in GTP results from the inhibition by these drugs of inosine monophosphate dehydrogenase (IMPDH), the first enzyme specific for the de novo synthesis of GMP. By serial passage of SV in A. albopictus cells in the presence of 25 μM MPA, we have now isolated viral mutants which are highly resistant not only to MPA but also to Rbv and TDA. For example, whereas 500 μM Rbv reduced the plaquing efficiency of SV STD by at least 10 6-fold, the the same concentration of Rbv reduced the plaquing efficiency of the MPA-resistant mutants less than 5-fold. This is the first example of a viral mutant resistant to the antiviral compound Rbv.

Nucleoside and nucleobase transport and metabolism in wild type and nucleoside transport-deficient Aedes albopictus cells

Nucleoside and nucleobase transport and metabolism were measured in ATP-depleted and normal Aedes albopictus mosquito cells (line C-7-10) by rapid kinetic techniques. The cells possess a facilitated diffusion system for nucleosides, which in its broad substrate specificity and kinetic properties resembles that present in many types of mammalian cells. The Michaelis-Menten constant for uridine transport at 28°C is about 180 μM. However, the nucleoside transporter of the mosquito cells is resistant to inhibition by nmolar concentrations of nitrobenzylthioinosine and the cells lack high affinity nitrobenzylthioinosine binding sites. The cells also possess an adenine transporter, which is distinct from the nucleoside transporter. They lack, however, a hypoxanthine transport system and are deficient in hypoxanthine phosphoribosyltransferase activity, which explains their failure to efficiently salvage hypoxanthine from the medium. The cells possess uridine and thymidine phosphorylase activities and, in contrast to cultured mammalian cells, efficiently convert uracil to nucleotides. An adenosine-resistant variant (CAE-3-6) of the C-7-10 cell line is devoid of significant nucleoside transport activity but transports adenine normally. Residual entry of various nucleosides into these cells and of hypoxanthine and cytosine into wild type and mutant cells is strictly non-mediated. The rate of permeation of various nucleosides and of hypoxanthine into the CAE-3-6 cells is related to their hydrophobicity. Uridine permeation into CAE-3-6 cells exhibits an activation energy of about 20 kcal/mol. At high uridine concentrations permeation is sufficiently rapid to partly overcome the limitation in nucleoside salvage imposed by the nucleoside transport defect in these cells.

Pleiotropic changes in cycloheximide-resistant insect cell clones.

Somatic cell mutants resistant to drugs that interact with the eukaryotic ribosome provide a useful tool for studies on ribosome structure, function, and genetics. From Aedes albopictus (mosquito) cells, cycloheximide-resistant mutants (Cx-705 and Cx-738) that were about 30-fold more resistant to cycloheximide than the parental cells have been obtained. The observation that protein synthesis in cell-free lysates from Cx-705 and Cx-738 cells was resistant to cycloheximide led us to suspect that the alteration in these mutants might affect the ribosome. The present studies show that the cycloheximide-resistant cells grow poorly and eventually die at 34.5 degrees C, a temperature at which wild-type cells grow normally. Relative to control cells, the cycloheximide-resistant cells show increased sensitivity to G-418, another antibiotic that interacts with the eukaryotic ribosome. However, there were no differences between cycloheximide-resistant cells and wild-type cells in sensitivity to puromycin, emetine, or cryptopleurine. Cx-705 cells were predominantly diploid; in contrast, the frequency of tetraploid nuclei in Cx-738 cells was about 40%.

Topological Mapping of Unique Epitopes on the Dengue-2 Virus NS1 Protein Using Monoclonal Antibodies

Monoclonal antibodies were produced against two distinct Thai dengue-2 (DEN-2) virus strains isolated in 1980 from dengue haemorrhagic fever patients. Nine of 36 hybridomas produced monoclonal IgG antibodies which reacted in radioimmune precipitation assays with the NS1 non-structural protein (42,000 mol. wt.) from DEN-2-infected C6/36 (Aedes albopictus) cells. The virus specificity of NS1-reactive monoclonal antibodies was determined by indirect immunofluorescence assays using LLC-MK2 cells infected with either the Thai 1980 DEN-2 isolates, prototype DEN viruses (four serotypes), Japanese encephalitis (JE), Murray Valley encephalitis, West Nile, Wesselbron or Tembusu viruses. Eight of the monoclonal antibody preparations were DEN-2-serotype specific. One preparation defined a special serological relationship between DEN-2 and JE viruses. Four preparations had detectable complement fixation titres using Thai DEN-2 virus antigen. Six spatially unique epitopes were identified using competitive binding assays.

Semliki Forest virus-induced polykaryocyte formation is an ATP-dependent event.

SummaryInfection of Aedes albopictus cells with Semliki Forest virus (SFV) leads to polykaryocyte formation below pH 6.2. This syncytium formation is accompanied by a decrease of the cellular ATP level. Addition of inhibitors of oxidative phosphorylation leads to a rapid, total depletion of ATP in infected cells at pH 6 and results in an inhibition of polykaryocyte formation. However, when cells were exposed for only a few minutes to pH 6 in the presence of the inhibitors and then kept at pH 7.2, the ATP level partially recovered to values sufficient for syncytium formation. Similar results were obtained after ATP depletion induced by 2-deoxyglucose. Thus, it can be concluded that SFV-induced syncytium formation is an ATP-dependent event.

Ultrastructural studies of Kunjin virus-infected Aedes albopictus cells.

Ultrastructural changes in Aedes albopictus cells infected with Kunjin virus were characterized from 12 to 72 h post-infection. Early in infection (16h), there were no prominent ultrastructural changes except for an increase in the number of vacuoles in the cytoplasm. As the infection progressed the rough endoplasmic reticulum appeared to lengthen and whorls of fibres were observed within some vacuoles. Virus particles were observed in small numbers scattered in the cytoplasm between 24 to 30 h after infection. However, by 48 h, large numbers of morphologically mature virus particles were visible, usually in association with the rough endoplasmic reticulum, with the clusters of vesicles or with convoluted smooth membranes. Although no definite evidence of precursor particles or of budding of virions was obtained, the clusters of vesicles and the various membranous structures appeared to play a role in the morphogenesis of this virus. The results are compatible with the hypothesis that maturation of virus particles occurs within all these virus-induced structures.