A convenient in vitro bioassay based on semiautomated quantification of live-cell reduction of tetrazolium dyes--3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-corb oxanilide sodium salt (XTT)--to formazan was developed and used to evaluate cytotoxic effects of two commercial insecticides (BT) derived from Bacillus thuringiensis subsp. kurstaki (Btk). Comparison of two target insect cell lines MG1 (Trichoplusia ni, cabbage looper midgut) and Sf9 (Spodoptera frugiperda, fall army worm oocyte) revealed similar cell-dependent responses in mitochondrial-associated electron transport activity. The 50% inhibition of formazan production (ID50) obtained by exposing these cells to 20 microM 2,4-dinitrophenol or 5 mM sodium azide occurred in the range 10(-7)-10(-6) International Units (IU) of BT cell(-1) 24 h(-1). Damage to cell adhesion and cytoarchitecture, revealed by light and electron microscopic analysis, increased with BT exposure and dose. MTT was superior to XTT as a cytotoxic indicator in kinetic studies related to spores, a major component of BT. Unless blocked by antibiotic (gentamicin), vegetative growth resulting from spore germination was the major cause of toxicity. The ID50 exposure time using vegetative Btk cells was approximately 0.1-0.2 times that required for BT spores, with or without intact parasporal proteins present. This difference in exposure is an indirect measure of the time required for spores to germinate and produce vegetative cells. The assay methodology developed here, if linked with suitable target and non-target animal cell types, should have broad application for conducting standardizable estimates of cytotoxic potential of any microbe-based biotechnology product.

Bacillus thuringiensis↗

PubMedDate not supplied

Plaque assay and replication of Tipula iridescent virus in Spodoptera frugiperda ovarian cells.

A plaque assay was developed for the study of Tipula iridescent virus (TIV) replication using a cell line derived from the fall army worm Spodoptera frugiperda (Sf9). Infection and plaque formation were monitored with time by phase contrast microscopy, video and fluorescent light microscopy. Structure of virions, viroplasmic centres and organelles of infected cells were examined by transmission electron microscopy (TEM). After 4 h postinfection, plaques were visibly detected within the cell monolayer by the presence of localized cell damage and production of numerous vesicular-like cytoplasmic structures. Quantitation of virions present per A260 unit of TIV preparation was determined by TEM. The number of visible plaques corresponded to virus concentration and 1 A260 produced approximately 10(5) plaques. DNA hybridization analysis revealed no gross differences in genomic DNA from TIV propagated in either Sf9 cells or wax moth Galleria mellonella larvae. These findings indicate that Sf9 is permissive for replication of TIV and superior by some parameters to other cell lines currently in use for the study of host cell/TIV interactions.

Animals↗

PubMedDate not supplied

Characterization of iridovirus IV1 polypeptides: mapping by surface labelling.

A comprehensive index of IV1(Tipula iridescent virus, or TIV)-associated polypeptides has been established using enrichments of "empty" and "filled" virions that are considered to be maturation-phase-related. The mapping strategy which involved one- and two-dimensional polyacrylamide gel electrophoresis, silver staining, disulphide bond reduction and 125I iodination of putative surface proteins revealed 103 and 116 polypeptides for empty and filled capsids, respectively. These estimates could be reduced to < or = 70 by reclassing multicharged polypeptides of approximately identical masses as single entitles. At least 10 polypeptides from empty and filled virions were involved in intermolecular sulphhydryl linkages and another 11 species were identified as putative outer shell (surface) polypeptides. These data provide useful criteria for iridovirus classification and identification of candidate polypeptides involved in capsid formation and maturation.

Animals↗