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.

Detection of Phthorimaea operculella granulovirus in vivo and in vitro by a specific DNA probe.

In analyzing populations of non-infected potato tuber moth (PTM) Phthorimaea operculella, using a total DNA probe from Phthorimaea operculella granulovirus (PhopGV), false positive reactions were obtained indicating homology between cellular and viral DNAs. Using a cloned 2.1 kbp fragment of PhopGV DNA, a specific digoxigenin-labeled probe was developed. This fragment did not show homology using both dot and Southern blot hybridization with either the genome of the larvae or genomes of the cell lines derived from the insect. The PhopGV-specific DNA probe detected as little as 1 ng, while the total DNA probe could detect even 35 pg of purified viral DNA. The 2.1 kb probe was highly specific for PhopGV. It gave negative results with two other granuloviruses isolated on Sesamia cretica and Spodoptera littoralis. The availability of a PhopGV-specific probe is an important prerequisite of detection of early stages of virus infection both in vivo on P. operculella larvae and in vitro on established P. operculella cell lines.

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.

Genetic heterogeneity of Phthorimaea operculella granulovirus: restriction analysis of wild-type isolates and clones obtained in vitro.

Genetic heterogeneity of a wild-type granulovirus (Tunisia isolate) of the potato tuber moth Phthorimaea operculella (Phthorimaea operculella granulovirus, Phop GV) has been studied. The heterogeneity was indicated by the presence of several submolar fragments in the profiles obtained by use of several restriction endonucleases. It was also demonstrated by variations in the restriction profile of the wild-type Tunisia isolate that had underwent since 1991 in our laboratory numerous passages in vivo. A comparison of the Tunisia isolate used in Egypt in the biological control programme with other PhopGV isolates indicated that it could not be related to any of the 3 genotypes previously defined. Five clones obtained from the Tunisia isolate in vitro were further grown both in vitro and in vivo. The restriction analysis of these clones demonstrated that none of them was identical to the parental wild type virus and to any other PhopGV geographic isolates. Genotypic differences between the clones were also shown. A 19 kbp BamHI fragment absent in the original Tunisia isolate but present in its passages since 1995 at a submolar concentration, was always present at a molar concentration in its clones. The presence of this fragment reflects probably a selection of one or more variants present in the original isolate and its possible adaptation to the growth in our laboratory conditions.

Evidence for two small viruses persistently infecting established cell lines of Phthorimaea operculella, deriving from embryos of the potato tuber moth.

Two small viruses were isolated from established cell lines of P. operculella deriving from embryos. The first one probably related to the Nodaviridae family, is a 30 nm in diameter icosahedral virus, with a bisegmented RNA genome and a single polypeptide of 39 kilodaltons. The second one related to the Parvoviridae family, is a 25 nm in diameter icosahedral virus with a DNA genome and a capsid constituted of 4 polypeptides of respectively, 90,000; 64,000; 56,000 and 43,500 daltons. The two viruses probably chronically infect the cell lines and may be consider latent viruses.

Yield and activity of Autographa californica multicapsid nucleopolyhedrovirus and Phthorimaea operculella granulosis virus in cloned and uncloned cell lines of P. operculella.

Three selected uncloned Pop 2, Pop 3, Pop 4 and two cloned cell lines Pop cl1A and Pop cl2B were derived from the original cell line established from Phthorimaea operculella (ORS-Pop-93). Three new non-selected cell lines ORS-Pop-94A, ORS-Pop-94B and ORS-Pop-95 were also established from embryos of the same insect. Differences in morphology, growth rate and polypeptide profile were determined between these cell lines. All the cell lines were susceptible to the Autographa californica nucleopolyhedrovirus (AcMNPV). The cloned cell lines produced higher levels of AcMNPV (TCID-50 and PIB) than the parental cells and at the same rate as the Sf9 reference cell line. Substantial amounts of viral DNA were synthesized in the clone Pop cl 2B after infection with the granulosis virus of the potato tuber moth P. operculella (PTMGV) and a complete multiplication was obtained in the ORS-Pop-95 cell line. The comparison between Pop cell lines which support limited or complete replication of certain baculoviruses can offer insights into some of the molecular barriers which restrict the host range of these viruses. These cell lines with variable susceptibility to baculoviruses could also be used for in vitro recombinations, increasing their virus host range to be used for the control of this pest.

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella.

A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19 degrees C) using modified Grace's medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19 degrees C and 38 h at 27 degrees C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19 degrees C. As long as the cells were maintained at 19 degrees C, virus multiplication could also be obtained at the same rate at 27 degrees C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.

Multiplication of Spodoptera littoralis granulosis virus in a cell line established from Phthorimaea operculella.

A complete replication of the Spodoptera littoralis granulosis virus (SpliGV) was obtained, in vitro by both virus infection and DNA transfection in the ORS-Pop-95 (Pop-95) cell line established from embryonic cells of the potato tuber moth, Phthorimaea operculella. SpliGV multiplied significantly during several passages in Pop-95 cells at 19 degrees C. When the cells were infected and kept at 19 degrees C for the first 4 hrs and then at 27 degrees C for the rest of the experiment (20 days), the viral multiplication proceeded at the same rate. Comparison of SpliGV progenies, multiplied either in vivo or in vitro, using electron microscopy and restriction profile analysis, showed their identity.

A new small RNA virus persistently infecting an established cell line of Galleria mellonella, induced by a heterologous infection.

A persistent infection in a Galleria mellonella cell line was revealed when infected with a maize stem borer picorna-like virus isolated on Sesamia cretica (MSBV). The new virus, completely different from the MSBV, is designated as G. mellonella cell line virus (GmclV), induces spectacular cytopathic effects, and is also considered efficient in vivo. The GmclV is a 29-nm-diameter isometric virus, with single-strand RNA of 2.9 x 10(6) Da molecular weight with a poly(A) tract. Its capsid is constituted of only two major polypeptides, of 34,500 and 32,500 Da, and no minor bands could be detected. The characteristics of the GmclV do not permit us to classify it with assurance. Even though it has not yet been identified as a picornavirus, it can be classified in the small RNA virus group of the Picornaviridae. G. mellonella represents a very interesting model, owing to the fact that two different persistent viruses belonging to the same family were isolated in vivo and in vitro, to further the understanding of the general phenomenon of persistency and induction.

Establishment of a cell line derived from embryos of the potato tuber moth phthorimaea operculella (Zeller).

A cell line from the main insect pest of potatoes in tropical and subtropical areas, Phthorimaea operculella (Zeller), was obtained from embryoculture. These cells were cultured in Grace's modified medium. The cell line, designated ORS-Pop-93, had a heterogeneous population consisting of spherical and spindle cells with great capacity to adhere and a doubling time of 40 h. They were subcultured for more than 60 passages. Their polypeptidic profile was different from profiles of other lepidopteran cell lines. The cell line supports the multiplication of the Autographa californica nuclear polyhedrosis virus.

Restriction map of the Casphalia extranea densovirus genome.

A physical map of the Casphalia extranea densovirus genome (CeDNV) was constructed. The size of the intact viral genome was estimated to be 4.9 kilobases or 1.6 MDa (single strand). The double-stranded CeDNV genomic DNA was cleaved with 26 restriction endonucleases and 20 restriction sites were mapped on the genome. The CeDNV DNA restriction map was compared to those of other densoviruses. Southern blotting hybridization experiments failed to reveal any homology between the genomes of CeDNV and Junoniacoenia densovirus (JcDNV).

Expression of epidermal differentiation antigens in cultures of interspecific somatic hybrids (human keratinocytes X mouse fibroblasts 3T3-4E).

Interspecific somatic hybrids have been prepared by fusion of human epidermal cells with mouse fibroblasts 3T3-4E using PEG 4000. Expression of epidermal differentiation antigens (bullous pemphigoid antigens, BP, keratin subsets 55-57 k and 67 k), markers of basal and suprabasal cells, were studied by immunocytochemistry for 10 passages. These markers were detected in the hybrids early after fusion, indicating that cells from both compartments were able to fuse with 3T3-4E cells. However, the hybrids expressing high molecular weight keratins were no longer detected after 7 days in primary cultures and serial passages, whereas those expressing BP antigens and vimentin persisted. Low molecular weight keratins 52 K and 50 K were detected by SDS-PAGE at the second passage in precipitates formed between labeled hybrid lysates and total keratin rabbit antiserum. Karyotype analysis showed mainly murine chromosomes and a submetacentric human chromosome between the 6th and the 10th passage.

[Interspecies somatic hybrid cultures from human epidermal cells and 3T3-4E mouse fibroblasts]. / Culture d'hybrides somatiques interspécifiques à partir de cellules épidermiques humaines et de fibroblastes de Souris 3T3-4E.

Interspecific somatic hybrids were obtained by fusion of adult human epidermal cells with Mouse fibroblasts 3T3-4E, deficient in thymidine kinase. These hybrids were identified by their morphology and by the presence of markers from the parental cells. Some characters of keratinocytes such as keratin subunits 50 and 51 K were present in primary cultures and disappeared after serial passages, whereas bullous pemphigoid basement membrane zone antigens persisted for at least 20 passages. At the 7th passage, a metacentric chromosome, and more often a submetacentric chromosome, presumably of human origin, were observed in some cells.

Identification of shared antigenic determinants of different polypeptides from Davis and Towne cytomegalovirus strains with monoclonal antibodies.

Six colonies producing antibodies were obtained by fusing mouse myeloma SP2-O cells with spleen cells from mice immunized with cytomegalovirus Davis strain. Among 88 surviving clones, 29 produced antibodies detectable by immunofluorescence on infected MRC5 cells and 2 others produced neutralizing antibodies against a homologous virus. Supernatants from these 31 positive clones and 4 others which were negative in immunofluorescence or neutralization were tested for their capacity to bind polypeptides from labelled Davis-infected cell extracts. Only 8 were found to be positive: five clones (A8, E3, F2, F8 and F20) precipitated 76 K, 60 K and 54 K bands; three others (A4, B9 and C24) precipitated 76 K and 54 K only. Surprisingly, these 8 monoclonal antibodies recognized a unique polypeptide 67 K of Towne-infected MRC5 cells. No correlation was found between (1) the pattern of fluorescence on MRC5 cells infected with Davis strain, (2) the neutralizing activity, and (3) the polypeptides recognized by the monoclonal antibodies.

Antigenic polypeptides of 3 reference strains recognized by sera from cytomegalovirus (CMV) infected patients.

The polypeptides of Cytomegalovirus (CMV) from 3 references strains (Davis, Ad 169 and Towne) were precipitated by immune sera from 5 renal transplanted patients with CMV-re-infection and 2 sera from patients with primary infection (one renal transplant and one child). Two antibody negative sera (tested in complement fixation test and immunofluorescence) were used as controls. The patterns of antigenic late polypeptides depended on the kinetics of in vitro infection. They differed between the 3 reference strains, and, for each strain, between the immune sera used for precipitation. Only 6 major bands were recognized by all sera in all reference strains. These results demonstrated he heterogeneity of CMV antigenic polypeptides responsible for antibody production in individual patients.