[Relation of insects to tobacco mosiac viruses].
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Identification and characterization of the repetitive DNA element in the genome of insect iridescent virus type 6.
The genome of the Chilo iridescent virus (CIV) was analyzed for existence of repetitive DNA sequences by DNA-DNA hybridization using a defined and complete gene library of the viral genome (209 kbp) and by heteroduplex mapping. These experiments revealed the presence of repetitive DNA elements in the CIV genome, which are located in the EcoRI fragment H and in the EcoRI DNA fragment C at the coordinates 0.535 to 0.548 (EcoRI/Pstl DNA fragment, 2.7 kbp) and 0.920 to 0.944 (PvuII CIV DNA fragment L, 5.1 kbp), respectively. The DNA nucleotide sequence (2708 bp) of the EcoRI/Pstl subfragment was determined. The comparative analysis of the DNA sequences of this particular region of the viral genome with the DNA sequences of the PvuII DNA fragment L (5064 bp) revealed the presence of several DNA sequences within the EcoRI/Pstl subfragment of the EcoRI CIV DNA fragment H which show homology to DNA sequences of the PvuII DNA fragment L. For example, a DNA element (box A, 91 bp) is located at nucleotide positions 1981 to 2072 of the EcoRI CIV DNA fragment H which are complementary (greater than 90%) to the nine regions of the PvuII DNA fragment L (L-boxes 1 to 9). Furthermore heteroduplex mapping revealed the existence of a stem-loop structure (stem, 65 +/- 10 bp and loop, 652 +/- 80 bp) at the genome coordinates 0.571 to 0.582 (2.5 kbp, HindIII/EcoRI subfragment of the EcoRI CIV DNA fragment H). This indicates that an inverted repeat sequence is located at this region of the viral genome. The DNA nucleotide sequence of this subfragment was determined (2555 bp) which confirmed the data obtained from electron microscopy. An inverted repeat DNA sequence located at nucleotide positions 304 and 1011 is able to form this type of stem-loop structure.
Base Sequence↗
Killing of flies in electrocuting insect traps releases bacteria and viruses.
Electrocuting insect traps (EIT) are popular devices frequently used by homeowners and food handlers attempting to localize the control of flying insects, including the ubiquitous house fly (Musca domestica L.). The traps contain a visual attractant and a high-voltage metal grid. Upon contact with the grids, the insects are disintegrated by the high voltage. As part of a systematic evaluation of EITs and their role in infectious disease spread, we quantitated spread of bacteria and a bacterial virus during electrocution of house flies. We loaded flies with Serratia marcescens or with the Escherichia coli phage PhiX174 and placed sprayed or fed flies into a room containing an EIT. While flies were being electrocuted, liberated particles and bacteria were assayed via agar plates or via air filtration samplers. Sprayed flies released one of every 10,000 of the added bacteria or viruses, and fed flies released one of every 1,000,000 of the consumed bacteria or viruses. Results of our studies suggest EITs could play a role in the spread of infectious disease agents, but the potential is influenced by the insect's route of contamination.
Animals↗
Weather, host and vector--their interplay in the spread of insect-borne animal virus diseases.
The spread of insect-borne animal virus diseases is influenced by a number of factors. Hosts migrate, move or are conveyed over long distances: vectors are carried on the wind for varying distances in search of hosts and breeding sites; weather and climate affect hosts and vectors through temperature, moisture and wind. As parasites of host and vector, viruses are carried by animals, birds and insects, and their spread can be correlated with the migration of hosts and the carriage of vectors on winds associated with the movements of the Intertropical Convergence Zone (ITCZ) and warm winds to the north and south of the limits of the ITCZ. The virus is often transmitted from a local cycle to a migratory cycle and back again.Examples of insect-borne virus diseases and their spread are analysed. Japanese, Murray Valley, Western equine, Eastern equine and St Louis encephalitis represent viruses transmitted by mosquito-bird or pig cycles.THE AREAS EXPERIENCING INFECTION WITH THESE VIRUSES CAN BE DIVIDED INTO A NUMBER OF ZONES: A, B, C, D, E and F. In zone A there is a continuous cycle of virus in host and vector throughout the year; in zone B, there is an upsurge in the cycle during the wet season, but the cycle continues during the dry season; there is movement of infected vectors between and within zones A and B on the ITCZ and the virus is introduced to zone C by infected vectors on warm winds; persistence may occur in zone C if conditions are right. In zone D, virus is introduced each year by infected vectors on warm winds and the arrival of the virus coincides with the presence of susceptible nestling birds and susceptible piglets. The disappearance of virus occurs at the time when migrating mosquitoes and birds are returning to warmer climates. The virus is introduced to zone E only on occasions every 5-10 years when conditions are suitable. Infected hosts introduced to zone F do not lead to circulation of virus, since the climate is unsuitable for vectors. Zones A, B and C correspond to endemic and zones D and E to epidemic conditions.Similar zones can be recognized for African horse sickness, bluetongue, Ibaraki disease and bovine ephemeral fever - examples of diseases transmitted in a midge-mammal cycle. In zones A and B viruses are transported by infected midges carried on the wind in association with the movement of ITCZ and undergo cycles in young animals. In these zones and in zone C there is a continual movement of midges on the warm wind between one area and another, colonizing new sites or reinforcing populations of midges already present. Virus is introduced at times into fringe areas (zones D and E) and, as there is little resistance in the host, gives rise to clinical signs of disease. In some areas there is persistence during adverse conditions; in others, the virus is carried back to the endemic zones by infected midges or vectors.Examples of viruses maintained in a mosquito/biting fly-mammal cycle are Venezuelan equine encephalitis and vesicular stomatitis. These viruses enter a migratory cycle from a local cycle and the vectors in the migratory cycle are carried over long distances on the wind. Further examples of virus spread by movement of vectors include West Nile, Rift Valley fever, yellow fever, epizootic haemorrhagic disease of deer and Akabane viruses.In devising means of control it is essential to decide the relationship of host, vector and virus and the nature of the zone in which the area to be controlled lies. Because of the continual risk of reintroduction of infected vectors, it is preferable to protect the host by dipping, spraying or by vaccination rather than attempting to eliminate the local population of insects.
African Horse Sickness↗
Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel group of insect-infecting RNA viruses.
The complete genome sequence of acute bee paralysis virus (ABPV) was determined. The 9470 nucleotide, polyadenylated RNA genome encoded two open reading frames (ORF1 and ORF2), which were separated by 184 nucleotides. The deduced amino acid sequence of the 5' ORF1 (nucleotides 605 to 6325) showed significant similarity to the RNA-dependent RNA polymerase, helicase, and protease domains of viruses from the picornavirus, comovirus, calicivirus, and sequivirus families, as well as to a novel group of insect-infecting RNA viruses. The 3' ORF2 (nucleotides 6509-9253) was proposed as encoding a capsid polyprotein with three major structural proteins (35, 33, and 24 kDa) and a minor protein (9.4 kDa). This was confirmed by N-terminal sequence analysis of two of these proteins. The overall genome structure of ABPV showed similarities to those of Drosophila C virus, Plautia stali intestine virus, Rhopalosiphum padi virus, and Himetobi P virus, which have been classified into a novel group of picorna-like insect-infecting RNA viruses called cricket paralysis-like viruses. It is suggested that ABPV belongs to the cricket paralysis-like viruses.
Amino Acid Sequence↗
Comparison of a DNA-DNA dot-blot hybridisation assay with light microscopy and radioimmunoassay for the detection of a nuclear polyhedrosis virus.
A dot-blot hybridisation assay was developed for the detection of a nuclear polyhedrosis virus (NPV) and was compared to light microscopy and radioimmunoassay (RIA). Using cloned NPV DNA labelled with 32P as a probe, a number of hybridisation assay procedures was examined. The assay was found to be more sensitive than differential staining, phase-contrast microscopy, or indirect solid-phase RIA with as few as 20 occlusion bodies (150 pg DNA) being detected. Samples do not require prior purification or DNA extraction. The assay was shown to be specific for NPV and has the potential to detect and discriminate between strains of the virus. With little modification the assay may be used to detect other insect viruses.
Animals↗
Autographa californica nuclear polyhedrosis virus efficiently enters but does not replicate in poikilothermic vertebrate cells.
The host range of the insect virus Autographa californica nuclear polyhedrosis virus (AcMNPV) was examined. AcMNPV could not initiate a productive infection in frog, turtle, trout, or codling moth cell lines. After exposure to AcMNPV, neither viral DNA nor RNA synthesis could be detected in these cell lines as assayed by nucleic acid probe hybridization. Entry of AcMNPV nucleocapsids into the cytoplasm and viral DNA into the nucleus, however, was as efficient in the nonpermissive cell lines as it was in a permissive insect cell line. The data suggest that the block in AcMNPV infection in these nonpermissive cell lines is at a stage subsequent to viral DNA entry into the nucleus.
Animals↗
Manawatu virus: a nodavirus isolated from Costelytra zealandica (white) (Coleoptera: Scarabaeidae).
An insect virus, called Manawatu virus (MwV), was isolated from a larva of the New Zealand grass grub Costelytra zealandica (White) (Coleoptera: Scarabaeidae). MwV was serologically related, but not identical, to several insect nodaviruses. The single capsid protein of MwV was 40,000 MW, the same as black beetle virus (BBV), but virus particles had a different electrophoretic mobility from BBV. The bipartite RNA genome, like other nodaviruses, consisted of two species of MW 1.1 and 0.46 million. MwV particles sedimented at 142 S and had an estimated density in neutral CsCl of 1.366 g/ml compared with 1.352 g/ml for BBV. The serological and physico-chemical properties, compared with other nodaviruses, indicate that MwV is unique.
Animals↗
Specific packaging of nodaviral RNA2 requires the N-terminus of the capsid protein.
Flock house virus (FHV), a member of the family Nodaviridae, is a nonenveloped, icosahedral insect virus whose capsids are assembled from 180 copies of a single type of coat protein. The viral genome is split between two segments of single-stranded positive-sense RNA, RNA1 and RNA2, which are packaged into a single virion. We previously demonstrated that synthesis of FHV coat protein in the baculovirus expression system results in assembly of virus-like particles whose capsids are indistinguishable from those of native virions, although the encapsidated RNA represents primarily cellular RNA. In contrast, expression of a deletion mutant lacking N-terminal residues 2-31 results in formation of multiple types of particles which differ in size, shape, and RNA contents. We postulated that the polymorphism was imposed by the type of RNA that the coat protein selected for packaging. In the current study we tested this hypothesis by analyzing the assembly of the mutant coat protein in Drosophila cells in the presence of replicating FHV RNAs. As anticipated, the resulting particles had the same shape and dimensions as wt virions. Surprisingly, however, they contained little RNA2 while packaging of RNA1 was not affected. Small amounts of defective interfering RNAs, which emerged rapidly in the presence of the mutant coat protein, were also detected. Taken together, these observations confirm our earlier hypothesis that selection of nonviral RNAs for packaging can significantly alter the assembly process. In addition, they demonstrate that the N-terminus of the FHV coat protein contains important determinants for recognition and packaging of RNA2. Our results provide the first evidence that encapsidation of the two genomic RNAs occurs independently and that the coat protein uses different regions for the recognition of RNA1 and RNA2.
Amino Acid Sequence↗
Electron microscopic observation of a newly isolated flavivirus-like virus from field-caught mosquitoes.
Of many unidentified virus strains which were isolated from field-caught mosquitoes by using C6/36 cells (a virus-sensitive clone of Aedes albopictus cells), three strains which formed small size plaques (SP virus) in C6/36 cells were investigated by electron microscopy. Although the SP virus strains did not react with antisera against known arboviruses in serological tests, they closely resembled flaviviruses in morphology. However, when they were compared to Japanese encephalitis (JE) virus, several differences in morphogenesis were observed. Proliferating membranous structures and electron-dense amorphous areas involving precursors of the virus were observed only in cells infected with the SP virus strains. Enlarged areas of endoplasmic reticulum containing mature virions were often observed adjacent to these structures. Since the SP virus strains were isolated from wild mosquitoes and multiplied only in mosquito cells, it seems appropriate to classify them as insect viruses which resemble togaviruses morphologically.
Aedes↗
Comparative properties of the inclusion body proteins of the nucleopolyhedrosis viruses of Neodiprion sertifer and Lymantria dispar.
A comparison was made on the properties of the inclusion body proteins of two insect viruses: the nucleopolyhedrosis viruses of the European pine sawfly, Neodiprion sertifer, Geoffroy, and the gypsy moth, Lymantria dispar, Linnaeus. The inclusion body proteins were characterized by the following parameters: amino acid composition, polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate--mercaptoethanol, isoelectric focusing, and alkaline protease activity. The properties of the inclusion body proteins of the two viruses were similar in many respects, but clear differences were observed. A principal difference was the absence of alkaline protease activity associated with the inclusion body proteins of N. sertifer nucleopolyhedrosis virus.
Amino Acids↗
Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector.
The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has played a major role in studies on the molecular biology of insect DNA viruses. Recently, this system has been effectively adapted as a highly efficient vector in insect cells for the expression of several mammalian genes. A cDNA sequence of the influenza (fowl plague) virus haemagglutinin gene has been inserted into the BamHI site of the pAc373 polyhedrin vector. Spodoptera frugiperda cells were co-transfected with this construct, pAc-HA651, and authentic AcNPV DNA. Recombinant virus was selected by adsorption of transfected cells to erythrocytes followed by serial plaque passages on S. frugiperda cells. We have determined the site of insertion of the haemagglutinin gene into the AcNPV genome by restriction enzyme cleavage and Southern blot hybridization analyses using haemagglutinin cDNA as a probe. The influenza haemagglutinin gene is located in the polyhedrin gene of AcNPV DNA. Immunofluorescent labelling, immunoprecipitation and immunoblot analyses with specific antisera revealed that S. frugiperda cells produce immune reactive haemagglutinin after infection with the recombinant virus. The haemagglutinin is expressed at the cell surface and has haemolytic capacity that has been activated by post-translational proteolytic cleavage. When chickens were immunized with S. frugiperda cells expressing haemagglutinin, they developed haemagglutinin-inhibiting and neutralizing antibodies and were protected from infection with fowl plague virus. These observations demonstrate that the haemagglutinin is processed in insect cells in a similar fashion as in fowl plaque virus-infected vertebrate cells and that it has full biological activity.
Amino Acid Sequence↗
Granulosis virus deoxyribonucleic acid: a closed, double-stranded molecule.
Studies on deoxyribonucleic acid purified from the granulosis virus of Trichoplusia ni have revealed the presence of a closed, double-stranded superhelix which sediments at 95S relative to relaxed circles (74S) and linear (60S) forms. Molecular weight estimates show that this insect virus deoxyribonucleic acid has a size of 100 x 10(6) daltons.
Centrifugation, Density Gradient↗
Persistent Hz-1 virus infection in insect cells: evidence for insertion of viral DNA into host chromosomes and viral infection in a latent status.
Persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. In this study, the long-term association between the Hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. With the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. Once viral reactivation takes place, the host cell dies. The persistently infected cells contain various amounts of viral DNA, and, in an extreme case, up to 16% of the total DNA isolated from infected cells could be of viral origin. Both pulsed-field gel electrophoresis and in situ hybridization experiments showed that some of these viral DNA molecules are inserted into the host chromosomes but that the rest of viral DNA copies are free from host chromosomes. Thus, Hz-1 virus is the first nonretroviral insect virus known to insert its genome into the host chromosome during the infection process. These data also suggest that the previously described persistent infection of Hz-1 virus in insect cells should be more accurately referred to as latent viral infection.
Animals↗
The expression of the Autographa californica nuclear polyhedrosis virus genome in insect cells.
This report presents a synopsis of recently published work in our laboratory on the molecular biology of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The following studies have been summarized. (1) On the mode of transcription of the AcNPV genome in insect cells. (2) Translation of proteins encoded in the 81.2 to 85.0 map unit segment of AcNPV. (3) Inserts of insect cell DNA in the AcNPV genome. (4) Expression of influenza (fowl plague) virus haemagglutinin in Spodoptera frugiperda insect cells, and successful immunization of chickens. (5) Synthesis of the influenza virus haemagglutinin in insect larvae by recombinant AcNPV. This insect virus system will continue to serve as a model for research on the molecular biology of insects. Moreover, the baculovirus system has been recognized as a very efficient and safe eukaryotic expression vector.
Animals↗
Increased insecticidal effect by a recombinant baculovirus carrying a synthetic diuretic hormone gene.
Genetically engineered insect viruses may be potential alternatives to conventional chemical insecticides. A recombinant gene for the diuretic hormone of the tobacco hornworm, Manduca sexta, has been introduced into a baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV). When silkworm larvae were injected with the resulting recombinant BmNPV, the diuretic hormone was expressed, causing a strong alteration in larval fluid metabolism. The recombinant virus killed infected larvae about 20% faster than the original virus.
Amino Acid Sequence↗
The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures.
Various cytopathological structures, known as inclusion bodies, are formed upon infection of cultured leafhopper cells by Rice dwarf virus, a member of the family Reoviridae. These structures include tubules of approximately 85 nm in diameter which are composed of the nonstructural viral protein Pns10 and contain viral particles. Such tubular structures were produced in heterologous non-host insect cells that expressed Pns10 of the virus. These tubules, when associated with actin-based filopodia, were able to protrude from the surface of cells and to penetrate neighboring cells. A binding assay in vitro revealed the specific binding of Pns10 to actin. Infection of clusters of cells was readily apparent 5 days after inoculation at a low multiplicity of infection with the virus, even in the presence of neutralizing antibodies. However, treatment of host cells with drugs that inhibited the elongation of actin filaments abolished the extension of Pns10 tubules from the surface of cells, with a significant simultaneous decrease in the extent of infection of neighboring cells. These results together revealed a previously undescribed aspect of the intercellular spread of Rice dwarf virus, wherein the virus exploits tubules composed of a nonstructural viral protein and actin-based filopodia to move into neighboring cells.
Actins↗
Analysis of RNA packaging in wild-type and mosaic protein capsids of flock house virus using recombinant baculovirus vectors.
Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two positive-sense RNA molecules, RNA1 (replicase gene) and RNA2 (coat protein gene), which are encapsidated into a single virion. Expression of coat protein in Sf21 cells using a baculovirus vector results in formation of virus-like particles (VLPs) whose capsids are structurally indistinguishable from native virions. However, RNA packaging is not specific for RNA2, the coat protein message. Using ribonuclease protection assays, we showed that the fraction of RNA2 in VLPs is 19% relative to the amount present in a population of native virions. To investigate possible reasons for the reduced level of RNA2, we generated two new baculovirus vectors, AcR1delta and AcR2delta, expressing the replicase gene and the coat protein gene, respectively. The inserted genes carried the self-cleaving hepatitis delta ribozyme sequence at the 3' end to allow for synthesis of RNA1 and RNA2 transcripts with authentic 3' ends. Infection of Sf21 cells with AcR2delta yielded VLPs that contained 66% RNA2 relative to native virions. Coinfection of Sf21 cells with AcR1delta and AcR2delta launched self-directed FHV replication and resulted in formation of particles most of which contained RNA1 and RNA2. However, a small fraction of particles containing cellular RNA was detected as well. The latter particles could be eliminated by infecting Sf21 cells with AcR1delta followed by transfection with in vitro synthesized transcripts of RNA2. We have further utilized this system to show that two coat protein deletion mutants with distinct RNA packaging defects form mosaic virus capsids but do not complement each other to rescue specific packaging of FHV RNAs.
Animals↗