Aedes Albopictus Cell Line Research Articles

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.

Insect chromosome isolation: a method for metaphase chromosome harvest from cultured cells of the mosquito Aedes albopictus.

A modified technique for bulk isolation of metaphase chromosomes from an in vitro cell line of the mosquito Aedes albopictus (Skuse) is described.

Selective disappearance of two secreted host proteins in the course of Semliki Forest virus infection of Aedes albopictus cells.

One secreted host protein of molecular weight 54,000 (SP 54) disappeared (from 24 to 48 h after infection) in Semliki Forest virus-infected Aedes albopictus cell clone C6/36 grown in both Mitsuhashi-Maramorosch basal medium and tissue culture medium 199 and reappeared when cells went into the permanently infected state. C6/36 is a high virus producer showing a cytopathic effect. A second secreted host protein of molecular weight 62,000 (SP 62) was prominent if cell clone C6/36 was grown in tissue culture medium 199. After infection in this medium, the protein showed a behavior similar to that described for SP 54. These secreted proteins were not affected in two original Aedes albopictus cell lines. SP 54 and SP 62 are monomeric proteins and structurally not related.

Characterization of invertebrate cell lines

A reliable, simple method for the separation of four cellular isozymes utilizing celluloseacetate electrophoresis is described. Cell extracts of 16 cell lines were examined previously, utilizing starch gel electrophoretic techniques. These same cell extracts were retested for their isozyme phenotype using the cellulose-acetate electrophoretic system. These data indicate that the results of the two electrophoretic systems are comparable. Isozyme analyses of freshly prepared cell extracts of the Aedes albopictus (ATC-15) and Spodoptera frugiperda (IPLB-SF-21AE) cell lines resulted in identical isozyme mobilities when compared with their respective stored counterparts. Since the cellulose-acetate electrophoretic system was able to separate cellular isozymes into characteristic patterns, distinctions between cell lines were made. The application of this technique for identification and characterization of invertebrate cell lines is discussed.

Phenotypic characterization of ouabain‐resistant Aedes albopictus cells

The phenotype of a ouabain-resistant Aedes albopictus cell line has been partially characterized. Treatment of ouabain-sensitive cells with 0.005-1.0 mM ouabain resulted in an 80% reduction in the uptake of 86rubidium (86Rb+), an ion with an affinity for the K+ pump binding site; ouabain-resistant cells showed only a 40% reduction with 1.0 mM ouabain. When ouabain-sensitive cells were incubated in the presence of ouabain (0.1 mM) for one and one-half to three hours, the molar ratio of intracellular Na+/K+ rose from 0.2 to 4.2. In ouabain-resistant cells, a similar treatment had very little effect. Based on [3H] ouabain-binding studies, ouabain-resistant cells were estimated to have 60% fewer binding sites per cell than ouabain-sensitive cells. The spontaneous mutation rate from ouabain sensitivity to ouabain resistance was calculated to be 1-6 x 10(-8) mutations/cell/generation, a value similar to that reported for mammalian cells at the analogous locus.

A mutant of chikungunya virus isolated from a line of Singh's Aedes albopictus cells by plaque formation on virus-sensitive cloned cells obtained from another Singh's A. albopictus cell line

A chikungunya (CHIK) virus-resistant line of Singh's Aedes albopictus (SAAK) cells released spontaneously two kinds of plaque-forming agents which could be distinguished by the plaque size on a sensitive clone C6/36 of another A. albopictus cell line (SAAR). Release of the large plaque (Lp) and the small plaque (sp) viruses into the culture medium occurred at different rates when the SAAK cells were subcultured. The Lp virus was removed from the SAAK cells by subculturing or cloning in the presence of anti-CHIK serum. Such Lp(-) cells continued to produce the sp virus, but were permissive for CHIK virus. The plaque-purified Lp virus, but not sp virus, interfered with the growth of CHIK virus in the C6/36 cells. The Lp virus resembled in physicochemical and serological properties CHIK virus grown in C6/36 cells. However the Lp virus was not pathogenic for suckling mice, was heat labile, and showed higher plating efficiency on the C6/36 cells than on BHK21 cells. The data indicate that the Lp virus is a mutant of CHIK virus and is responsible for the CHIK virus resistance of the SAAK cells.

Proteins specified by togaviruses in infected Aedes albopictus (Singh) mosquito cells.

Yields of greater than 10(7) p.f.u./ml at 28 or 37 degrees C of the alphavirus Sindbis and the flavivirus Kunjin were obtained in the Aedes Albopictus (Singh) cell line, the latent periods being 4 to 6 and 10 to 12 h, respectively. Despite a high background of host protein synthesis, virtually all the virus-specified proteins of the flaviviruses Kunjin, Dengue-2 and Japanese encephalitis were labelled and resolved by slab gel electrophoresis of infected and uninfected cell proteins. In contrast, only one induced protein, of mol. wt. 30 000, was identified in cells labelled during Sindbis virus infection. The envelope glycoprotein V3 of Kunjin virus was resolved as a double band in samples of infected cytoplasm labelled with 3H-glucosamine, similar to that of carbohydrate-labelled V3 in vertebrate (Vero) cells. Attempts to reduce host protein synthesis selectively during labelling periods were unsuccessful using either a hypertonic inhibition block or treatment with 0.1 mu g actinomycin D per ml. The most efficient labelling of Kunjin virus-specified proteins was achieved at 37 degrees C in the presence of actinomycin D. The largest non-structural flavivirus protein NV5 migrated slightly faster than NV5 from infected vertebrate (Vero) cells. The small non-structural proteins NV1, NV1 1/2 and NV2 from infected mosquito cells were successively trimmed during post translational periods exceeding 70 min, compared to much shorter periods reported previously for post translational modifications of these proteins in vertebrate cells.

Plaque Production by Arboviruses in Singh's Aedes albopictus Cells

We report plaquing tests of 124 virus strains, mostly arboviruses of 21 serological groups, in Singh's line of Aedes albopictus cells. Thirty of these plaqued: all were arboviruses of six groups and were known or presumed to be mosquito borne. Failing to plaque were 86 strains of arboviruses, mostly tick borne, two strains of insect pathogens, and six animal viruses not classified as arboviruses. Among mosquito-borne agents, plaquing ability appeared related to serological classification. California group and most A-group viruses failed to plaque, but nearly all members of B and Bunyamwera groups readily plaqued. Within serological group B, 14 of 16 mosquito-borne agents plaqued, but none of 13 tick-borne or vector-unassociated viruses did so. Some implications of these results for recognition and classification of arboviruses are discussed.

Plaque Production by Arboviruses in Singh's Aedes albopictus Cells

We report plaquing tests of 124 virus strains, mostly arboviruses of 21 serological groups, in Singh's line of Aedes albopictus cells. Thirty of these plaqued; all were arboviruses of six groups and were known or presumed to be mosquito borne. Failing to plaque were 86 strains of arboviruses, mostly tick borne, two strains of insect pathogens, and six animal viruses not classified as arboviruses. Among mosquito-borne agents, plaquing ability appeared related to serological classification. California group and most A-group viruses failed to plaque, but nearly all members of B and Bunyamwera groups readily plaqued. Within serological group B, 14 of 16 mosquito-borne agents plaqued, but none of 13 tick-borne or vector-unassociated viruses did so. Some implications of these results for recognition and classification of arboviruses are discussed.

Studies of interferon production in Aedes albopictus mosquito cells.

SummarySingh's Aedes albopictus mosquito cell line was infected with Sindbis and West Nile viruses in order to assess the ability of these invertebrate cells to produce interferon. The viruses grew...

II. Multiplication of Chikungunya and O'nyong-nyong viruses in Singh's Aedes cell lines.

We have been interested in insect cells as a tool to throw some light on virus-vector relationships. In studies conducted during 1968 and 1969 (Buckley, 1969a and b), 32 strains of arboviruses were examined for their ability to infect Aedes cells. Twenty-seven viruses belonged to 9 different antigenic groups and 5 were ungrouped. The findings of Singh and Paul (1968a) that mosquito-borne viruses including chikungunya (CHIK) multiply either in both or at least in one of the cell lines were confirmed. However, we have found exceptions to the tentative generalization made by Singh and Paul that tick-borne arboviruses fail to infect the Aedes cells. In our hands, Colorado tick fever virus (CTF), an ungrouped tick-borne virus, multiplies in the Aedes albopictus cell line, but not in the A. aegypti cell line (Buckley, 1969a). These results confirm the findings of Yunker and Cory (1969). In addition to CTF, four viruses of the Kemerovo group, namely Kemerovo, Lipovnik, Tribec and Chenuda, non-group B tick-borne viruses, infected both Aedes cell lines (Buckley, 1969b). Tick-borne viruses of group B such as Kyasanur Forest disease (Singh and Paul, 1968 b), Langat (Buckley, 1969a) and Kadam (W. N. Mugo, personal communication) do not multiply in the Aedes cells, whereas 5/18 non-group B tick-borne viruses infect either one or both of Singh’S Aedes cell lines.