Development and characterization of a continuous cell line, AFKM-On-H, from hemocytes of the European corn borer Ostrinia nubilalis (Hübner) (Lepidoptera, Pyralidae).

The corn borer, Ostrinia nubilalis, is a very important pest in different countries, and the in vitro system of the insect could be a useful tool for isolation and characterization of the pathogens and physiological responses of the insect. In this context, a cell line was derived from the hemocytes of the European corn borer and was named AFKM-On-H for, respectively, O. nubilalis, Armand Frappier, King Mongkut Institutes, and Hemocytes. This cell line was initiated and maintained in Ex-Cell 400 medium supplemented with 10% heat-inactivated fetal bovine serum. The cells, mostly spherical in shape, not firmly attached to the plastic culture flasks, were passaged up to 200 times by repeated gentle pipetting of the cells. The doubling times at the 80th and 125th passages at 28 degrees C and at the 122th and 169th passages at 25 degrees C were 40, 29, 35, and 34 h, respectively. The AFKM-On-H cell line was further characterized by the morphology, karyotype, random amplified polymorphic DNA analysis, and isozyme profiles. Susceptibility of the cell line to cytoplasmic polyhedrosis viruses (CPV) Euxoa scandens (EsCPV), Dendrolimus punctatus (DpCPV), and Choristoneura fumiferana (CfCPV); nuclear polyhedrosis viruses [Autographa californica (AcMNPV) wild type and recombinant, Antherea yammamai (AnyaNPV)]; and Chilo iridescent virus was demonstrated. Relative sensitivities of the cell line to Bacillus thuringiensis and Metarhizium anisopliae toxins and effects of the molting hormone 20-hydroxyecdysone on this new hemocyte cell line were characterized.

Characterization of cell lines developed from the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae).

In order to isolate new pathogens (viruses, microsporidia, etc.) or to evaluate the efficiency of some pathogens (serovarieties and mutants of Bacillus thuringiensis, fungi, etc.) in the control of Colorado potato beetle, an economically important pest, we established four cell lines from tissues of this insect. One was initiated from embryonated egg fragments in the M3 medium supplemented with 20% fetal bovine serum (FBS) and then transferred after several passages to the Ex-Cell 400 medium with 20% FBS. Another was initiated from larval hemocytes in Ex-Cell 400 with 5% FBS. Finally, two other cell lines were initiated from adult hemocytes: one in the Ex-Cell 400 with 20% FBS and 1% of lipid mixture and the other in the Ex-Cell 400 with 5% FBS only. These cell lines have been characterized by their morphology with light and electron microscopy, their karyotypes, cell growth, and isozyme analysis. Each cell line differed in morphologic, karyologic, growth, and isozyme patterns. The cell line initiated from embryonated eggs was growing slower than the three initiated from hemocytes. The cytotoxicity of solubilized crystal delta-endotoxins from different B. thuringiensis formulations (M-One, Trident, MYX-1806, Teknar-HPD, and Thuricide) and of destruxins, mycotoxins from Metarhizium anisopliae, was tested on these cell lines. They are sensitive to the solubilized toxins of some strains of B. thuringiensis (serovar. San Diego and serovar. tenebrionis) and to destruxins, and they can be used for the bioassay and detection of toxins and for the study of the mechanism of their action on coleopteran cells.

DNA content analysis of insect cell lines by flow cytometry.

The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cell lines were stable among the passages and during the length of time culture. This technique was demonstrated to be useful for the detection of mixed cell lines and nucleopolyhedrovirus cell infection, using Autographa californica MNPV. The flow cytometry gives interesting results on the cell cycle and the ploidy level; it appears as a good tool for insect cell lines characterization.

Effects of bacillus thuringiensis and destruxins (Metarhizium anisopliae mycotoxins) combinations on spruce budworm (Lepidoptera: tortricidae)

Laboratory bioassays were performed in order to assess the efficacy of a combination of a commercial preparation of Bacillus thuringiensis var. kurstaki (B.t.k.) (Thuricide) with destruxins (Metarhizium anisopliae mycotoxins) on the fifth instar of Choristoneura fumiferana Clemens. Lethal doses were determined for each microbial agent and used as a basis for the bioassays involving a combination of the two agents. Interaction or the lack of interaction was determined by comparing the observed and the theoretically expected mortality rates. Seven out of 10 different combinations demonstrated synergism between the two agents. The modelization applied on results allowed us to establish the following general equation: LDmixture = 1.259(LDDx) + 1.129(LDBt) - 0.016(LDDx)(LDBt) + 12.196. Such an equation explains the relationship which exists between the two lethal agents (R2 = 0.99). Our results suggest that the two agents contribute to the synergism in the system and that a combination of both could be an efficient means of controlling C. fumiferana populations and of reducing the dose of B.t.k. which is usually required for such a control. Copyright 1998 Academic Press.

Characterizations of natural and induced polyhedrin gene mutants of Bombyx mori cytoplasmic polyhedrosis viruses.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) polyhedrin gene from natural mutant strain B, B2 and P was cloned and the amino acid sequence was compared with that of wild-type virus (strain H). Chimeric polyhedrin genes (8 types) containing only one site of mutation within the amino acid sequence were also constructed. Fifteen types of polyhedrin genes were introduced into a baculovirus expression vector and hexahedral, acicular, pyramidal, or amorphous polyhedra were formed in infected cells. These results demonstrated that the shape of polyhedra as well as the crystallization pattern of the polyhedrin could be changed by mutations at respectively N-terminal and C-terminal regions of BmCPV polyhedrin gene.

Effect of mutations on the intracellular localization of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin.

We have already cloned the polyhedrin genes of the wild-type strain H Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) and its mutant, strain A. In this work, polyhedrin genes of mutant BmCPV strains C1 and C2 were cloned and their nucleotide sequences were determined. The polyhedrin amino acid sequences of strains C1 and C2 were compared with that of strain H. Strains C1 and C2 contained two and three sites of mutation in their polyhedrin genes, respectively. Four amino acids (249RLLV) were added at the carboxy terminus of the polyhedrin of strain A, C1 and C2 and the corresponding polyhedrin genes were introduced into a baculovirus expression vector. Intracellular localization of expressed polyhedrin as well as the morphology and localization of polyhedra were investigated by Western blot and microscopy analysis. Recombinant baculovirus containing the polyhedrin gene of strain H produced hexahedral polyhedra in both the cytoplasm and the nucleus. However, the hexahedral polyhedra of strain A were localized only in the nucleus. Normal polyhedra were not observed in cells infected with recombinant baculoviruses expressing strain C1 or C2 polyhedrin genes, but amorphous structures were found in infected cells. Results of expression of a chimaeric luciferase-containing carboxyl-terminal sequence of strain A demonstrated that this sequence was responsible for the nuclear localization. We suggest that a mutation at the carboxy terminus of BmCPV polyhedrin led to nuclear localization of polyhedrin and that several other mutations were responsible for modification of the crystallization pattern of polyhedrin.

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

Three cell lines (A.t. GRIP-1, 2, and 3) were established from Aedes triseriatus (Say) embryonated eggs or neonate larvae and their morphology, growth, karyotype, and isozyme pattern were studied. The isozyme alleles observed in the 3 cell lines also were found in adults of the original mosquito colony. Each cell line differed in enzymatic, morphological, and karyotypical patterns. La Crosse encephalitis (LAC) and snowshoe hare (SSH) viruses, members of the California encephalitis virus group, were able to replicate in these 3 cell lines. Furthermore, these cell lines, especially A.t. GRIP-1, were more sensitive than the Aedes aegypti (L.) (ATC 10) cell line for detection of small amounts of delta-endotoxin of Bacillus thuringiensis serovar. israelensis (de Barjac).

Expression in Escherichia coli of the cloned polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus.

Cloned cDNA of genomic segment 10 of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was placed downstream from the lambda PL promoter in expression plasmid pRC23 and expressed in Escherichia coli cells. A polypeptide of the same molecular weight (28 kDa) as natural polyhedrin was synthesized at the level of approximately 10% of total host cell protein. This polypeptide was identified as CPV polyhedrin (r-polyhedrin) after comparative studies. The r-polyhedrin did not form any crystalline structure in E. coli cells but instead accumulated in the form of an insoluble inclusion body, even though natural polyhedrin is known to form a crystalline matrix (polyhedra) in infected insect cells. The purified r-polyhedrin complex, like natural polyhedra, was not soluble in neutral or acidic buffer but soluble in alkaline buffer. Upon solubilization, the r-polyhedrin complex did not undergo proteolytic degradation, while natural polyhedra were digested into small peptides by the associated protease. Incubation of r-polyhedrin with natural polyhedra in alkaline buffer, however, degraded the r-polyhedrin, resulting in an identical profile of peptide products to that of natural polyhedra. These results indicate that even though r-polyhedrin molecules produced in E. coli cells are not in the natural conformation, the molecules can present the identical cleavage sites to the polyhedra-associated alkaline protease. Experiments showed that the alkaline protease was associated with the matrix of polyhedra and not with virus particles.

A simplified method for the characterization of nuclear polyhedrosis virus genomes.

A simplified restriction endonuclease analysis procedure is described which allows the characterization of baculovirus DNA obtained directly from a single larvae without purification of virus. This rapid method was used to demonstrate the genomic stability of nuclear polyhedrosis viruses (NPVs) from Agrotis segetum, Euxoa messoria and Mamestra brassicae after several passages in Euxoa scandens.

Nucleotide sequence of the polyhedrin gene of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV).

The double-stranded RNA genome of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV) was reversely transcribed to the double-stranded DNA and cloned into pIBI30. The complete nucleotide sequence of cloned genome segment 10, which encodes the virus polyhedrin polypeptide, was determined. The EsCPV polyhedrin gene consists of 881 bp and possesses an open reading frame that codes for a polypeptide of 269 amino acids (MW 30.1K), consistent with an apparent MW of 30K determined by SDS-PAGE for purified polyhedrin. The sequence is identical to that reported for the amino terminus of polyhedrin from the CPV of Orgyia pseudotsugata; however, no amino acid or nucleotide sequence homology was found between the EsCPV polyhedrin and that from Bombyx mori CPV (BmCPV) or several nuclear polyhedrosis viruses. The hydrophilic profiles and predicted secondary structures of both EsCPV and BmCPV polyhedrin show some similarities, mainly in the amino half of the polypeptides. These data should be helpful in identifying the domains responsible for the polyhedrin crystallizing properties.

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.

Protein Synthesis in a Lymantria dispar 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.

Activity of California encephalitis group viruses in Entrelacs (province of Quebec, Canada).

During the summer of 1979, indicator rabbits were placed in three sites in Entrelacs (Laurentian area, province of Quebec) and mosquitoes were collected in order to monitor arbovirus activity in the area. Eight seroconversions to California encephalitis (CE) group viruses were detected in rabbits during June, July, and August. Twenty-five strains identified as members of the CE group were isolated: 3 were obtained from viremic rabbit sera, 1 from adult Aedes communis reared in the laboratory from field-collected larvae, and 21 from mosquito pools. Twenty-two of these were typed as snowshoe hare (SSH) virus. No evidence of La Crosse (LAC) virus was detected but three strains belonging to the CE group showed antigenic properties different from reference SSH, LAC, or Jamestown Canyon (JC) viruses. One isolate identified as Flanders virus was obtained from Culex pipiens. Three mosquito species (A. communis, A. punctor, and A. excrucians) were involved in the transmission cycle of SSH virus in Entrelacs. This is the first report, in the province of Quebec, of SSH isolation from animal sera and the first demonstration of its transovarial transmission.

Production of cytoplasmic polyhedrosis virus (CPV) polyhedra in a gamma irradiated Lymantria dispar cell line.

Lymantria dispar cells wee exposed to different doses of gamma radiation one hour after infection with cytoplasmic polyhedrosis virus (CPV). It was found that irradiated cells can produce infectious polyhedra. Modifications in the structure and in the process of maturation of the polyhedra were noted. The number of polyhedra per cell increased significantly after cell irradiation at 10(4) and 10(5) rads but no change was noted after cell treatment at 10(2) rads. On the other hand, morphological changes and a high mortality rate were noted in cell cultures treated at intensities higher than 10(3) rads. Therefore, the total yield of polyhedra produced when using 10(2) or 10(4) rads was similar to that obtained in normal cells but dropped significantly after cell irradiation at 10(5) rads.

Electrophoretic characterization of proteins and RNA of cytoplasmic polyhedrosis virus (CPV) from Euxoa scandens.

A cytoplasmic polyhedrosis virus (CPV) was isolated from Euxoa scandens and propagated in vitro in Lymantria dispar cells. The virions and polyhedra were analyzed by polyacrylamide gel electrophoresis. Polyhedra produced in vivo and in vitro contained six polypeptides with identical relative mobilities while nonoccluded viral particles produced in vivo were composed of four polypeptides. The RNA content of the purified virions extracted from infected midgut was resolved in ten segments with molecular weights similar to that of CPV type 5 RNA.

Titration of a cytoplasmic polyhedrosis virus by a tissue microculture assay: some applications.

A simple tissue microculture technique was developed for the titration of a cytoplasmic polyhedrosis virus (CPV) from Euxoa scandens. The procedure was similar to the 50% tissue culture infectious dose assay, but a single infected cell, detected by the presence of cytoplasmic polyhedra, was scored rather than the degeneration of cell monolayers. The filtration of CPV suspensions resulted in decreased virus titers under certain conditions. This microculture assay was used to determine the effect of cell disruption methods on virus yields. Sonication of infected cells was more efficient than freeze-thawing for the recovery of nonoccluded virus.

Comparative methods of titration of Silverwater virus, an arthropod borne virus.

Silverwater virus (SIL) was titrated in vivo and in vitro. Using a microculture technique, TCID50 was found to be much more sensitive than both the plaque forming assay and the LD50. The latter was the least sensitive mode of titration of Silverwater virus. The addition of DEAE dextran and trypsin to the viral inoculum increased the number and diameter of the plaques.

[Electron microscopy studies of the early stages of mosquito cell infection in vitro by the chilo iridescent virus and the infectious bovine rhinotracheitis (author's transl)]. / Premiers stades d'infection de cellules de moustiques in vitro par le chilo iridescent virus et le virus infectieux de la rhinotracheite bovine. Microscopie électronique

The early stage of the viral infection in vitro of mosquito cells were studied by electron microscopy using a microculture technique. We demonstrate in this paper that two viruses: iridescent (CIV) and herpetic (Infectious bovine rhinotracheitis) can enter by viropexis and be uncoated in several lines of mosquito cells.