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Queensland fruit fly virus, a probable member of the Picornaviridae.

A picornavirus was isolated from various life stages of the Queensland fruit fly, Dacus tryoni. This virus, Queensland fruit fly virus (QFFV) has virions with a diameter of 30 nm and a sedimentation coefficient of 178 S. One third of the particles in preparations were empty capsids or natural top component (NTC) with a sedimentation coefficient of 95 S. The buoyant density (rho) of virions and NTC in CsCl was 1.34 and 1.30 g/ml respectively; small amounts of a dense component (rho = 1.45 g/ml) were also detected. The capsid contained three major protein species of molecular weight (mol.wt.) 41,700, 36,500, and 31,300, in approximately equimolar proportions. NTC contained three major species of mol. wt. 44,700, 41,700, and 31,300. The nucleic acid present only in the bottom component virions was RNA and comprised about 30% of the particle weight and had a mol. wt of 2.88 kd, contained a poly(A) tract, and had a base ratio: G = 20; A = 32; C = 15; U = 33. The mol. wt. of the virion was estimated to be approximately equal to 9.5 kd. When virions were heated at 56 degrees C and above, they converted into artificial top component (ATC), which had the same protein composition as the virion when analysed by SDS-PAGE. In immunodiffusion tests the virions and NTC were indistinguishable, but a minor difference in antigenicity was detected between the virions and ATC. Virions were stable between pH 3 and 9 inclusive, and between 5 and 7 in the presence of 0.14 M NaCl. Immunodiffusion tests showed that QFFV was serologically unrelated to a range of picornaviruses as well as an unclassified virus isolated from the Mediterranean fruit fly, Ceratitis capitata. The data show that QFFV is probably a member of the Picornaviridae, genus Enterovirus.

Animals↗

Nucleotide sequence and genomic organization of a newly isolated densovirus infecting Dendrolimus punctatus.

The nucleotide sequence of a novel icosahedral DNA virus infecting Dendrolimus punctatus has been determined. The genome is 5039 nt long and includes inverted terminal repeats of 200 nt containing 131 nt long J-shaped terminal hairpins. The 'plus' strand of the genome contains three large open reading frames (ORFs), the left and the mid-ORFs (within the left ORF) in the left-half encoding the non-structural proteins and the right ORF in the right-half encoding viral capsid proteins. NS1 protein contains conserved replication initiation and DNA-dependent ATPase/helicase domains. VP1 protein contains a conserved PGY and phospholipase A2 motifs and shows high identities with VPs of Casphalia extranea densovirus and Bombyx mori densovirus-1 belonging to the genus Iteravirus. Phylogenetic analysis also revealed that this virus is most closely related to Casphalia extranea densovirus and Bombyx mori densovirus-1. Consequently, this virus was considered as a new third member of the genus Iteravirus of the subfamily Densovirinae, and designated Dendrolimus punctatus densovirus.

Animals↗

Virulence of cloned variants of Autographa californica nuclear polyhedrosis virus.

The relative virulence of five different genotypic variants of Autographa californica nuclear polyhedrosis virus was tested by determining the 50% lethal dose of occluded virus for larvae of Trichoplusia ni. The 50% lethal dose values of uncloned virus and the five cloned genotypic variants ranged between 10 and 21 polyhedra per larva, and no statistically significant differences were observed. Cloning has therefore neither enhanced nor decreased the virulence of this potential microbial pesticide.

Genes, Viral↗

Changes in nucleus and surface of Lepidoptera cells cultivated in vitro after infection with nuclear polyhedrosis viruses (Autographa californica).

Changes in cell nuclei after infection of Lepidoptera cells, demonstrated by means of the fluorochrome "33 258H", permit the observation of virus DNA synthesis in a light microscope. Parallel occurring changes in cell surfaces, analysed in a scanning electron microscope, also permit an exact differentiation between infected and non-infected cells.

Animals↗

The 5' noncoding region sequence of the Choristoneura biennis entomopoxvirus spheroidin gene functions as an efficient late promoter in the mammalian vaccinia expression system.

About 100 nucleotides of DNA sequence at the 5' noncoding region of the Choristoneura biennis entomopoxvirus spheroidin gene was chemically synthesized and inserted into a vaccinia expression vector, interrupting the vaccinia thymidine kinase gene. When the bacterial beta-galactosidase gene was introduced downstream of this sequence and a recombinant vaccinia virus containing these inserts was obtained by homologous recombination, beta-galactosidase was shown to be expressed at a high level late in the vaccinia infection cycle. The level of beta-galactosidase expression was four- to fivefold higher with this spheroidin-vaccinia recombinant virus than with a similar recombinant in which the beta-galactosidase gene was under the control of the vaccinia 7.5-kDa promoter. Primer extension and S1 mapping of the 5' terminus of the beta-galactosidase transcript located the transcription initiation site within the spheroidin DNA sequence, confirming the promoter nature of this DNA sequence in the vaccinia system. Dot blot analysis indicated that the difference in beta-galactosidase expression with these two recombinant viruses can be attributed to the difference in their transcript levels. We also demonstrated that full promoter activity encoded in the spheroidin 5' noncoding sequence was contained within a 38-nucleotide DNA fragment.

Animals↗

A baculovirus expression vector derived from the basic protein promoter of Autographa californica nuclear polyhedrosis virus.

The basic protein of Autographa californica nuclear polyhedrosis virus (AcMNPV) is associated with virus DNA in virion nucleocapsids and is produced in infected cells during the late phase of gene expression. A transfer vector was constructed containing the beta-galactosidase gene, under the control of a copy of the putative basic protein promoter, in place of the polyhedrin gene within the AcMNPV EcoRI fragment I. After cotransfection of Spodoptera frugiperda cells with the transfer vector and infectious AcMNPV DNA, polyhedrin-negative recombinant viruses were selected which expressed high levels of beta-galactosidase. Radiolabelling of infected cell proteins showed that beta-galactosidase was expressed at the same time as the viral basic protein, between 8 to 24 h post-infection, with a peak synthesis at 12 to 15 h. These results demonstrated that the temporal regulation of the basic protein promoter was not affected by its position within the virus genome. Furthermore, a new baculovirus vector system is now available for high level expression of foreign genes at earlier times in infected cells.

Animals↗

Sequence of RNA2 of the Helicoverpa armigera stunt virus (Tetraviridae) and bacterial expression of its genes.

The complete nucleotide sequence of RNA2 of Helicoverpa armigera stunt virus (HaSV), a member of the Tetraviridae, was determined by characterization of cloned cDNA and PCR products and direct sequencing of genomic RNA. The capped, positive sense, single-stranded RNA is 2478 nucleotides in length and has two overlapping open reading frames (ORFs) likely to be cistrons which are situated between terminal non-coding regions of 282 and 168 bases, 5' and 3', respectively. Extensive secondary structure of the RNA strand is indicated, including a tRNA-like structure at the 3' terminus which is the first such structure discerned in an animal virus. The first ORF encodes a 17 kDa PEST protein (p17) of unknown function while the second ORF encodes the 71 kDa coat protein precursor (p71) that is cleaved at an Asn-Phe site into the 64 kDa and 7 kDa coat proteins. The precursor coat protein is 66% identical to that of another tetravirus, the Nudaurelia omega virus, with most of the difference residing in a 165 amino acid region located in the middle of the sequence. Despite the extensive similarity, no serological relationship was observed between the two viruses, suggesting that the dissimilar region is exposed on the capsid exterior. Expression in bacteria of the two RNA2 gene products shows they are likely to be expressed by a leaky scan-through mechanism. Bacterial expression of p71 did not produce virus-like particles while expression of p17 produced large arrays of mostly hollow, hexagonal tube-like structures.

Amino Acid Sequence↗

Artifical transfer and morphological description of virus particles associated with superparasitism behaviour in a parasitoid wasp.

In parasitoids, the adaptive significance of superparasitism (laying of egg(s) in already parasitized hosts) has been the subject of strong controversy. The current view is to interpret this behaviour as an adaptation to increased competition for hosts, because the supernumerary egg still has a chance to win possession for the host. However, we recently discovered that in the solitary parasitoid Leptopilina boulardi, superparasitism is rather caused by an unknown infectious element: stable non superparasitizing lineages (NS) are transformed into stable superparasitizing lineages (S) after eggs from both lineages have competed inside the same host (superparasitism). In this report, we investigate the nature and location of the causative agent. Involvement of bacteria is unlikely because antibiotic treatments do not affect wasp phenotype and because bacterial 16S ribosomal DNA was not detected using PCR. We report successful injection experiments showing that the causative agents are located in wasp poison gland and ovaries and are stably inherited. Electron microscopic studies demonstrate that long filamentous virus particles located in wasp oviducts are strongly associated with superparasitism behaviour, leading to reconsider the adaptive significance of this behaviour in parasitoids. Interestingly, parasitoids are often infected with similar viruses for which no phenotypic effect has been documented. This raises the possibility that they could induce the same behavioural manipulation.

Animals↗

Baculovirus bioassay not dependent upon polyhedra production.

An immunoperoxidase-based quantitative assay has been developed for infectious units of Autographa californica nuclear polyhedrosis virus infecting TN-368 cells. Major benefits of this assay over existing quantitative assays for baculoviruses are that it is not dependent upon polyhedra production, it is clear-cut and easy to score, and results are obtained within 24 h.

Immunoenzyme Techniques↗

Replication and morphogenesis of Amsacta moorei entomopoxvirus in cultured cells of Estigmene acrea (salt marsh caterpillar).

A study of the sequence of morphogenic events occurring within Amsacta moorei entomopoxvirus-infected Estigmene acrea cells is presented. Stages in virion development, and the various cytopathic effects observed in these cells between 0 and 120 h post-infection (p.i.) are described. Events in the early stages of virion assembly (24 to 48 h p.i.), the formation of the outer viral membrane (48 to 72 h p.i.) and the development of occlusion bodies or spheroids (72 to 120 h p.i.) were identified. Cells grown in TC100 culture medium supported production of mature virus particles, the majority of which were either of the intracellular naked virion form, or mature virions incorporated into occlusion bodies. Only limited production of the extracellular enveloped form was observed in these cells.

Animals↗

A novel capsid expression strategy for Thosea asigna virus (Tetraviridae).

This paper presents evidence that Thosea asigna virus (TaV) has a unique capsid expression strategy and is a member of the Nudaurelia beta-like genus of the Tetraviridae. Electron microscopy of TaV particles indicated a 38 nm, T = 4 icosahedral capsid similar in structure to that of Nudaurelia beta virus (NbetaV). TaV particles have a buoyant density of 1.296 g/cm3 in CsCl and consist of two capsid proteins of 56 and 6 kDa. The virus genome contains a genomic RNA molecule of 6.5 kb and a subgenomic molecule of 2.5 kb. Northern blotting of TaV RNA indicated a genomic organization similar to that of NbetaV. The capsid gene of TaV is carried on both the genomic and subgenomic RNA molecules, while the RNA polymerase gene is present only on the genomic RNA. Cloning and sequencing of the TaV capsid gene identified an open reading frame that could potentially encode a capsid precursor protein of up to 82.5 kDa. The N-terminal sequences of the capsid proteins were compared with the nucleotide sequence of the capsid open reading frame. The sequences indicate that the pre-protein is cleaved at two positions to produce the 56 and 6 kDa capsid proteins as well as a predicted third protein that was not detected in the mature virion. Phylogenetic analysis of the capsid proteins indicated that TaV is more closely related to NbetaV than to the Nudaurelia omega-like viruses. The eight beta-sheets that make up a jelly roll structure in the TaV capsid protein were identified by computer analysis.

Amino Acid Sequence↗

A suggested taxonomy and nomenclature for the cricket paralysis and Drosophila C virus complex.

Biophysical, biological, and serological characters are presented which in combination allow isolates of cricket paralysis virus (CrPV) and Drosophila C virus (DCV) to be separated and seven distinct strains of CrPV to be defined. Reference isolates for CrPV and DCV are suggested along with a system of nomenclature that allows the passage history of a particular isolate to be easily described.

Animals↗

[The safety of the preparation viroden for vertebrate animals].

Toxicity-pathogenicity test of viroden, a new preparation, and its acting agent--a mosquito densonucleosis virus (MDV) has been carried out on warm-blooded animals. It is shown that the preparation is not toxic for laboratory animals (white common mice, rats, guinea pigs, rabbits), chicken embryos and cell cultures of warm-blooded animals. The MDV is not adapted to a warm-blooded organism with different ways of introduction and in passages. Using electron and luminescent microscopy, serological reactions, specific test systems and a biological test for sensitive insects no explicit or latent infection was found in animals, chicken embryos and cell cultures of vertebrates with primary infection and in passages. Sensibilized animals shown an immunological rearrangement of the organism proceeding by the retarded hypersensitivity type.

Animals↗

[Histopathological studies of silworms (Bombyx mori Linn.) spontaneously infected with nuclear polyhedrosis].

Pathohistological changes in silkworms, spontaneously affected by nuclear polyhedry are investigated. The J-124 X C-122 hybrid after the fourth moulting is tested. After being subjected to a low temperature stress the silkworms were killed and material for pathohistological studies were taken from them. It is established that the most suitable tissues for the replication of the polyhedry virus of the silkworm were the cells of the fat bodies, the tracheal matrix and the hypoderm. Not all polyhedra form simultaneously. The virus as polyhedral bodies is detected in the infected tissues, cells respectively, on the 55th day (at the earliest) by the action of the low temperature stress. The histopathological investigation is recommended as a method for reliable and relatively early diagnoses.

Animals↗

A Moloney murine leukemia virus-based retroviral vector pseudotyped by the insect retroviral gypsy envelope can infect Drosophila cells.

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. Previous data suggest that gypsy ENV-like ORF3 mediates viral infectivity. We have produced in the 293GP/LNhsp701ucL.3 human cell line a Moloney murine leukemia virus-based retroviral vector pseudotyped by the gypsy ENV-like protein. We have shown by immunostaining that the gypsy envelope protein is produced in 293GP/LNhsp701ucL.3 cells and that vector particles collected from these cells can infect Drosophila cells. Our results provide direct evidence that the infectious property of gypsy is due to its ORF3 gene product.

Amino Acid Sequence↗

In vitro translation of cricket paralysis virus RNA.

Cricket paralysis virus RNA acted as a messenger in a translation system and directed incorporation of 35S-methionine into protein. Polyacrylamide gel analysis of the proteins demonstrated the presence of proteins of comparable molecular weight to the viral structural proteins and also potential high molecular weight precursors.

Insect Viruses↗

Mass production and purification of Euproctis chrysorrhoea (L.) nuclear polyhedrosis virus.

The rearing of larval Euproctis chrysorrhoea (L.) on an artificial wheatgerm diet is described. The larvae grew well on the diet, but took over ten weeks to complete their development, reflecting their prolonged life-span in the field. Using these larvae it was possible to mass produce a nuclear polyhedrosis virus (NPV) isolated from this species, with a view towards its use as a biological insecticide. A mean of 5.86 x 10(9) semi-pure NPV polyhedral inclusion bodies/g virus-killed larva was produced. The purification method described (simple centrifugation) removed over 99% of contaminant urticaceous hairs. Further purification through a sucrose gradient removed more hairs, but also resulted in a loss of NPV of about 35%.

Animals↗