Infection In Drosophila Research Articles

Replicative and conservative transpositional recombination of insertion sequences.

We have presented the results of experiments with IS903- and IS10- derived transposons that have led us to the following conclusions: The predominant mechanism of transpositional recombination of these IS elements is a donor-suicide process that results intermolecularly in a simple IS insertion. This process presumably involves little or no replication of the IS. Intramolecular transposition by this process normally results in nonviable products. However, in the particular situation where the transpositional target lies within the transposon, viable products are obtained; these are deletions and deletion-inversions. Deletions between an IS and a target lying outside the element (the conventional "adjacent deletion") occur by a fully replicative process analogous to the formation of cointegrate molecules in intermolecular transposition. The ability of an IS to promote adjacent deletions correlates closely with its ability to fuse replicons into a cointegrate. Before transposition can occur, a complex of the transposase and both IS ends is probably formed. Requirement for such a pretranspositional complex is suggested by the effect on transpositional frequency of changing the distance between the ends. Our results do not support any of the asymmetrical models for transposition. They are, however, compatible with a modified version of the symmetric model proposed by Shapiro (1979). It is interesting to note the similarity between the structures generated by intramolecular simple transposition of an inverse transposon and the circular structures apparently formed by retroviral and copia autointegrative transposition. Shoemaker et al. (1981a,b) and Flavell and Ish-Horowicz (1983) have characterized circular molecules from retrovirally infected cells and Drosophila tissue-culture cells, respectively. The structures of some of the circular molecules resemble deletions and deletion-inversions (Fig. 3B). To our knowledge, a circular species containing two long terminal repeats (LTRs) and an adjacent deletion, which we predict could only occur by a fully replicative process given the similarity in geometry of an LTR to an IS, have not been found. It would appear, then, that the molecule containing two LTRs acts as an inverse transposon, integrating into itself. Shoemaker et al. (1981b) and Flavell and Ish-Horowicz (1983) have also suggested that these products arise from molecules containing two LTRs. We suggest that the two inside LTR ends interact in a conservative, intramolecular, simple transpositionlike event.

Characterization of cricket paralysis virus- and Drosophila C virus-induced RNA species synthesized in infected Drosophila melanogaster cells

The use of insects as food for humans or as feed for animals is an alternative for the increasing high demand for meat and has various environmental and social advantages over the traditional intensive production of livestock. Mass rearing of insects, under insect farming conditions or even in industrial settings, can be the key for a change in the way natural resources are utilized in order to produce meat, animal protein and a list of other valuable animal products. However, because insect mass rearing technology is relatively new, little is known about the different factors that determine the quality and yield of the production process. Obtaining such knowledge is crucial for the success of insect-based product development. One of the issues that is likely to compromise the success of insect rearing is the outbreak of insect diseases. In particular, viral diseases can be devastating for the productivity and the quality of mass rearing systems. Prevention and management of viral diseases imply the understanding of the different factors that interact in insect mass rearing. This publication provides an overview of the known viruses in insects most commonly reared for food and feed. Nowadays with large-scale sequencing techniques, new viruses are rapidly being discovered. We discuss factors affecting the emergence of viruses in mass rearing systems, along with virus transmission routes. Finally we provide an overview of the wide range of measures available to prevent and manage virus outbreaks in mass rearing systems, ranging from simple sanitation methods to highly sophisticated methods including RNAi and transgenics.

Isolation of a bisegmented double-stranded RNA virus from Thirlmere reservoir.

A novel bisegmented double-stranded RNA virus has been isolated from water processed from Thirlmere reservoir. The virus is icosahedral, 58 nm in diam., has a buoyant density of 1.32 g/ml in CsCl, has an S value of 400 and a RNA/protein ratio of 0.087. The two linear segments of RNA have approx. mol. wt. of 2.26 X 10(6) and 2.09 X 10(6). The virus contains six polypeptides. The virus was isolated in Drosophila melanogaster cells and fails to replicate in other insect, amphibian, avian, piscine, mammalian and plant cells tested. The virus is biochemically different from infectious pancreatic necrosis virus (IPNV) and Drosophila X virus (DXV). The virus is also serologically unrelated to IPNV (strain Sp) and another invertebrate pathogenic virus, Tellina virus 1. The virus shares common antigens with DXV but is not completely identical.

The proteins expressed by different isolates of Drosophila C virus.

Isolates of Drosophila C virus (DCV) from Drosophila flies obtained in geographically different regions were adapted to growth in Drosophila tissue culture cells. The viruses, purified from tissue culture cells, were shown to be serologically related to one of the isolates ("O" from Ouarzazate, Morocco). Analysis of the structural proteins by polyacrylamide gel electrophoresis demonstrated differences between the isolates. Labelling intracellular proteins of infected Drosophila melanogaster cells with 35S-methionine at 28 degrees C demonstrated the presence of the virus structural proteins and their immediate precursors. Raising the temperature to 37 degrees C both before and during the pulse period inhibited the processing of the high molecular weight proteins and resulted in a greater "shut-off" of host cell proteins than viral induced proteins. This allowed the precursor proteins to be compared as well as the structural proteins of the different strains. It was possible to clearly distinguish differences between the isolates on the basis of the induced proteins, although limited proteolysis of corresponding proteins showed marked similarities. Hence it is possible to distinguish between different isolates of the "same" small RNA-virus of insects from geographically different regions.

In vivo and in vitro synthesis of the proteins expressed by the RNA of black beetle virus.

Black beetle virus is a small RNA virus with a single capsid protein of molecular weight congruent to 40,000. Two intracellular proteins, a presumed polymerase (molecular weight 110,000) and a protein having a molecular weight slightly greater than the capsid, were observed when infected Drosophila cells were pulsed with 35S-methionine. Viral RNA coded for the synthesis of several major proteins with molecular weights between 110,000 and 38,000 in rabbit reticulocyte lysate. Long chases of the translated products demonstrated processing of capsid protein precursors into capsid protein. Partial proteolysis demonstrated similarities in the proteins synthesised in vitro and in vivo.

The intracellular proteins induced by cricket paralysis virus in Drosophila cells: the effect of protease inhibitors and amino acid analogues.

Treatment of Cricket paralysis virus infected Drosophila cells with iodoacetamide before radiolabelling with 35S-methionine results in the appearance of two high molecular weight polypeptides of approximately equal to 200,000 molecular weight, not apparent in untreated infected cells (17). To attempt to differentiate between the effects of iodoacetamide being attributable to either alteration of initial polyprotein or inhibition of the protease (either cellular or viral) the effects of a spectrum of protease inhibitors were examined. These included aprotinin, leupeptin, pepstatin, elevated zinc concentration, phenyl methyl sulphonyl-fluoride, N-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). TLCK and TPCK both inhibited the cleavage of proteins which demonstrates an inhibition of the protease activity. The introduction of amino acid analogues into the infected cells before pulsing also results in the appearance of higher molecular weight proteins. This could be attributed to alternation of the polyprotein making it nonsusceptible to digestion with pre-existing cellular protease or newly synthesized viral protease. The possibility that the presence of the amino acid analogues results in alteration of a viral coded protease cannot be eliminated.

Occurrence of antibodies against insect virus proteins in mammals: simple model to differentiate between passive exposure and active virus growth.

Antibodies against an "enterovirus-like" virus of insects, cricket paralysis virus, occur in the sera of domestic animals. When these antibodies were used in combination with the immunoprecipitation of radiolabeled virus proteins from infected Drosophila cells in culture, it could be demonstrated that the animals were exposed to preformed virus.

The Polypeptides Induced in Drosophila Cells by a Virus of Heteronychus arator

SUMMARY The virus isolated from the black beetle, Heteronychus arator, is an icosahedral ribovirus with a diam. of 30 nm and contains one major structural polypeptide of mol. wt. 40000. The virus can be grown in Drosophila melanogaster cells and also titred in this line using a plaque assay method. Infected Drosophila cells pulsed with 35S-methionine in the presence of actinomycin D induced three virus polypeptides of mol. wt. 110000, 40000 and 8000. Short pulses, chases with excess non-radioactive methionine and pre-pulse treatments with protease inhibitors failed to demonstrate processing of virus-induced polypeptides.

Drosophila X virus RNA polymerase: tentative model for in vitro replication of the double-stranded virion RNA.

An RNA polymerase activity was found to be associated with the infectious drosophila X virus particles extracted from infected flies. The rate of synthesis was at first linear as a function of time, and then a plateau was reached. During the linear phase of the synthesis, the template and product were associated as replicative intermediates which were larger than the double-stranded RNA of the drosophila X virus genome, but the final product of the reaction was indistinguishable from the RNA genome with respect to its density, sedimentation coefficient, electrophoretic mobility, and RNase resistance. The results indicated that both strands of the genomic RNA were copied. The implications of these findings with regard to other virion polymerases are discussed.

Contamination and persistent infection ofDrosophila cell lines by reovirus type particles

A new type of contaminant particles persistently infectedDrosophila cell lines. On an ultrastructural, morphogenetic and histochemical basis, they are similar to viruses of the Reoviridae group. They have been namedDrosophila K virus (DKV).

Isolation and Biological Properties of Drosophila X Virus

SUMMARY Drosophila X virus described here appeared as a contaminant in Drosophila melanogaster. It is pathogenic for the inoculated flies, inducing anoxia sensitivity and death in these insects. An assay based on these symptoms in flies has been developed. Immunofluorescence has been used to study the characteristics of infected Drosophila cell cultures. A permanent infection can be established in these cultures. This virus is morphologically similar to several ungrouped vertebrate and invertebrate viruses like IPNV, IBDV and Tellina tenuis virus. Its possible origin is discussed.

Herpetomonas megaseliae sp. n. (Protozoa: Trypanosomatidae) from Megaselia scalaris (Loew, 1866) Schmitz, 1929 (Diptera: Phoridae)

A new intestinal trypanosomatid was found parasitizing Megaselia scalaris. Forms with posterior kinetoplasts occurred in both natural infections and cloned cultures, but not in experimental infections. Average size and morphological variation were reduced in experimental infections with cloned stocks, compared to natural infections. A growth comparison was made between Herpetomonas muscarum and H. megaseliae. Differences in day of peak population, maximum numbers, and total time of survival were observed in culture. Attempts to infect Drosophila melanogaster with H. megaseliae were unsuccessful. The primary characteristic used to separate Herpetomonas from other genera of insect trypanosomatids is a posterior kinetoplast in up to 40% of culture forms (Rogers and Wallace, 1971). An intestinal trypanosomatid which exhibited a high percentage of posterior kinetoplasts in culture was discovered in Megaselia scalaris (Loew, 1866) Schmitz, 1929 (see Borgmeier, 1965, for synonyms). The organism was isolated and a study was made of its morphology in culture and in the insect host. In addition, a comparison with Herpetomonas muscarum was made of growth in culture. Morphological and host differences between the newly isolated trypanosomatid and published descriptions indicated that the parasite was an undescribed species of the genus Herpetomonas. The isolate was cloned and the description below is based on cloned material in culture and in experimental infections. MATERIALS AND METHODS Four culture media were used in the study: (1) Mansour's Medium (Dollahon and Janovy, 1971); (2) 1% proteose-peptone-glucose (PPG); (3) Wallace's Medium (Wallace and Clark, 1959); and (4) A medium consisting of 5 ml Locke's Solution (Tobie et al., 1950) into which one guinea pig fecal pellet was placed (LPG). The LPG was then autoclaved. The parasites were isolated from the larvae of M. scalaris into Mansour's Medium with 700 mg dihydrostreptomycin and 700 units procaine penicillin-G (Penstrep) per ml. The isolate was cloned 4 times, twice by serial dilution and twice Received for publication 4 January 1972. * This study was supported in part by a grant from the University of Nebraska Research Council. by isolation of a single organism with a capillary pipet. Each clone was made from a stock which had been previously cloned. Our present stock has thus been successively cloned 4 times. A combination of 4,000 units of procaine penicillin-G and 5 mg dihydrostreptomycin (Penstrep) per ml was used in the first transfer after the fourth cloning. A week-old culture in Wallace's Medium, a descendant of the final clone, was used to study the morphology and to describe the trypanosomatid in culture. Cultures were maintained at 25 C. H. muscarum, isolated from Phaenicia sericata, was obtained from Dr. F. G. Wallace, University of Minnesota. It was received in Wallace's Medium and subsequently maintained in our laboratory in Mansour's or Wallace's Medium. For growth studies, 20 ml of LPG, PPG, or Mansour's Medium was added aseptically to sidearm flasks. A flask of each medium was inoculated with 0.25 ml of a H. muscarum culture of the new trypanosomatid. Hemocytometer counts were made on the 3rd, 5th, 7th, and 10th days after inoculation. Zero time populations were calculated from hemocytometer counts of the inoculum. Colonies of uninfected host flies, M. scalaris (identification confirmed by Dr. William Robinson, Virginia Polytechnic Institute), were initiated with eggs gathered from guinea pig litter pans. Eggs were washed once in a 1:10,000 zephiran-chloride solution and once in distilled water. Washed eggs were placed in 8-oz cotton-gauze-stoppered Frenchquartered bottles containing 50 ml of a Drosophila medium (Strickburger, 1967) which had been modified by the substitution of 250 ml of autoclaved, ground Purina Guinea Pig Chow for 38 ml dried yeast. Uninfected fly colonies were periodically checked for infection. Colonies of infected flies were initiated with larvae from guinea pig litter pans. Drosophila melanogaster, Oregon-R strain, were obtained from Dr. D. D. Miller, University of Nebraska, and were maintained in the same fly medium. The Drosophila were not found to be naturally infected with trypanosomatid parasites. Insects were infected with culture forms con-