Establishment and characterization of three cell lines from Aedes triseriatus (Diptera : Culicidae)

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DNA content analysis of insect cell lines by flow cytometry

Le contenu en ADN de plusieurs lignées cellulaires a été déterminé par cytométrie de flux. Les profils de 8 lignées testées sont différents, caractérises par la présence de plusieurs pics (2 à 7) correspondant aux différents niveaux de ploïdie. Deux lignées apparaissant constitutées de 2 populations cellulaires bien distinctes (Cf124 et BmN). Les profils obtenus sont stables au cours des passages et durant la culture. Cette technique permet de détecter des mélanges de lignées mais aussi des niveaux d'infections virales en utilisant un nucléopobyhediomes de #Autographa californica$ (AcMNPV). La cytométrie de flux donne des renseignements intéressant sur le cycle cellulaire et le niveau de ploïdie des cellules d'insectes et apparaît comme un bon outil de caractérisation. (Résumé d'auteur

Molecular cloning and characterization of cytoplasmic polyhedrosis virus polyhedrin and a viable deletion mutant gene

The double-stranded RNA genome of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was converted to double-stranded DNA and cloned into plasmid pBR322. The complete nucleotide sequence of cloned genome segment 10, which encodes virus polyhedrin polypeptide, was determined. The CPV polyhedrin gene consists of 942 based pairs and possesses a long open reading frame that codes for a polypeptide of 248 amino acids (molecular weight, 28,500), consistent with an apparent molecular weight of 28,000 previously determined for purified polyhedrin. No sequence homology was found between CPV polyhedrin and polyhedrins from several nuclear polyhedrosis viruses. In addition to the polyhedrin gene, we completed the sequence analysis of a small deletion mutant gene derived from the polyhedrin gene. This mutant gene consists of two subset domains of the polyhedrin gene, i.e., the 5'-terminal 121 base pairs and the 3'-terminal 200 base pairs. An in vitro transcription demonstrated that the small mutant gene is transcribed by virion-associated RNA polymerases. These data confirm the importance of CPV terminal sequences in virus genome replication.</jats:p

Protein Synthesis in a <i>Lymantria dispar</i> Cell Line Infected by Cytoplasmic Polyhedrosis Virus

The efficiency of replication of a cytoplasmic polyhedrosis virus isolated from a member of the order Lepidoptera, Euxoa scandens, was studied in eight different lepidopterean cell lines. Lymantria dispar cells, which were found to support viral replication, more efficiently, were used to follow the kinetics of appearance of viral-specific polypeptides by a 2-h pulse with [ 35 S]methionine. Five polypeptides (ca. 120,000 molecular weight [120K], 105K, 66K, 46K, and 28K) were identified as components of the polyhedral inclusion bodies, and two polypeptides (112K and 39K) were assigned as viral-particle polypeptides. All these polypeptides were present after 24 h and were still being produced 96 h after infection. The rate of synthesis of the major polyhedral polypeptide (28K) increased in the time course of infection, whereas the background of cellular polypeptides seemed to be unaffected. An indirect immunoperoxidase technique, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was blotted to a nitrocellulose membrane, showed that traces of the major polyhedral polypeptide were found from 8 h postinfection. </jats:p