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[Biological activity of viral RNA in nuclear polyhedroses of lepidoptera].
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[Aphid-transmissible viruses and the helper viruses (author's transl)].
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POSSIBLE INTEGRATION OF VIRAL NUCLEIC ACID INTO THE GENOME OF ANIMAL CELLS.
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Determination of the oral susceptibility of South African livestock-associated biting midges, Culicoides species, to bovine ephemeral fever virus.
A total of 10 607 Culicoides midges (Diptera: Ceratopogonidae) were fed on either sheep or horse blood containing not less than 6.5 log10 TCID50/ml of bovine ephemeral fever virus (BEFV). Insects were collected during two consecutive summers from two distinct climatic areas. Two seed viruses, originating from either South Africa or Australia, were used separately in the feeding trials. Blood-engorged females were incubated at 23.5 degrees C for 10 days and then individually assayed in microplate BHK-21 cell cultures. Of the 4110 Culicoides that survived, 43% were C. (Avaritia) imicola Kieffer and 27% were C. (A.) bolitinos Meiswinkel. The remainder represented 18 other livestock-associated Culicoides species. Although BEFV was detected in 18.9% of midges assayed immediately after feeding, no virus could be detected after incubation. The absence of evidence of either virus maintenance or measurable replication suggests that most of the abundant livestock-associated Culicoides species found in South Africa are refractory to oral infection with BEFV. Future studies should be carried out using species of mosquitoes that are associated with cattle in the BEF endemic areas.
The estimation of virus density in isopycnic cesium chloride gradients.
Methods for estimating the density of virus particles after isopycnic centrifugation in cesium chloride gradients were examined. If solutions were prepared in different buffers, the standard tables relating refractive index and density, based on solutions prepared in water, did not give consistent estimates even if corrections were made for the greater refractive indices of the buffers. Depending on the method and buffer used, estimates of the density of cricket paralysis virus, an insect picornavirus, differed by as much as 0.05 g/ml. However, consistent estimates were obtained if separate regression equations were derived to relate refractive index and density for each different buffer solution of cesium chloride. This has wider implications and could account for density differences or variations in estimates reported for many other viruses.
Expression of biologically active middle T antigen of polyoma virus from recombinant baculoviruses.
Two different recombinant baculoviruses have been generated for expressing the middle T antigen (MT) of polyoma virus in insect (Sf9) cells. One (pAcI-PyMT) produces moderate levels of MT and the other (pVL-PyMT) high levels. Indirect immunofluorescence and cellular fractionation studies with pAcI-PyMT infected Sf9 cells give results similar to those observed with wild type polyoma virus infected mouse cells, and show MT to be mainly associated with cytoplasmic membranes in the insect cell. In the latter, a sub-population of MT is phosphorylated in in vitro protein kinase assays. The yields of MT from pVL-PyMT infected cells are high enough to suggest that this protein can now be produced by this method in sufficient amounts for definitive biochemical and crystallographic analyses.
Synthesis of the complete 200K polyprotein encoded by cowpea mosaic virus B-RNA in insect cells.
The coding sequence for the entire 200K polyprotein of cowpea mosaic virus (CPMV) B-RNA was expressed in insect cells by using baculovirus expression vectors. The 200K polyprotein, which harbours all virus functions required for RNA replication, is completely cleaved into 170K and 32K products by the 24K protease activity contained within the polyprotein. Further processing of the 170K protein into CPMV-specific products of 60K, 84K, 87K, 110K and 112K occurred to a limited extent, similar to that observed in cowpea cells. Electron microscopy of insect cells in which the 200K protein was produced revealed the presence of membranous vesicles and electron-dense structures which were not seen in cells infected with wild-type baculovirus. Similar cytopathic structures develop in the cytoplasm of CPMV-infected cowpea cells and are thought to be the site of membrane-bound viral RNA replication. The electron-dense structures in insect cells could be preferentially labelled with several CPMV-specific antisera and Protein A-gold. Since electron-dense structures were not observed in cells in which the 170K protein only was produced, it seems that the 32K protein has a role in keeping the B-RNA-encoded proteins in these structures together. Membranous vesicles were also observed in insect cells in which the 60K protein only was produced. Use of specific antibodies and Protein A-gold showed that the 60K protein is associated with these vesicles, indicating that the 60K protein may induce the formation of vesicles. Although proteolytic processing of the 200K polyprotein and the induction of cytopathic structures indicate that the CPMV proteins produced in insect cells are functional, it has not been possible to demonstrate RNA polymerase activity in extracts of these cells using an oligo(U)-primed assay. The results indicate that in the assay an additional component is lacking and/or that the CPMV polymerase is not able to start RNA synthesis on an exogenous template.
SUSCEPTIBILITY OF AN INSECT TISSUE CULTURE TO INFECTION BY VIRUS PREPARATIONS OF THE NUCLEAR POLYHEDROSIS OF THE SILKWORM (BOMBYX MORI L.).
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Immunization of cotton rats with the human respiratory syncytial virus F glycoprotein produced using a baculovirus vector.
The F glycoprotein of respiratory syncytial virus in insect cells was produced using a baculovirus expression vector to examine its potential as a subunit vaccine. Two different forms of the F glycoprotein were expressed: the intact F (F) glycoprotein and the truncated (Ft) glycoprotein, in which the COOH-terminal anchor region was deleted. The F glycoprotein remained cell associated, whereas the Ft glycoprotein was secreted into the media of infected cells. In contrast to the processing of the F0 precursor into its F1 and F2 subunits that was observed in mammalian cells, a second cleavage site within the F1 subunit was recognized by the insect cell proteases and resulted in the formation of two F1 subunits. The baculovirus-expressed Ft glycoprotein induced neutralizing antibodies in cotton rats and protected vaccinated animals from challenge with respiratory syncytial virus.
Characterization of an iridescent virus isolated from the velvetbean caterpillar, Anticarsia gemmatalis.
An insect iridescent virus has been isolated from diseased velvetbean caterpillars, Anticarsia gemmatalis, found in Argentina. The cytopathology is similar to that reported for other iridescent viruses with infected larvae exhibiting blue-purple opalescence. Icosahedral particles (n = 119) purified by sucrose gradient rate zonal centrifugation had dimensions of 145 +/- 7 nm (point-to-point) and 136 +/- 12 nm (side-to-side). The sedimentation coefficient was 2250 +/- 10 S20,w when the virus was suspended in phosphate buffer. Characterization of proteins by SDS-polyacrylamide gel electrophoresis revealed 24 polypeptides with a single major species of 53.6 kDa. Purified viral DNA had a density of 1.6902 g cm-3 in equilibrium ultracentrifugation, corresponding to a guanine:cytosine content of 32.2%. Analysis of digests of this DNA with two restriction enzymes revealed an average molecular size of 181.8 +/- 5.6 kbp. A 499-bp fragment was amplified from the genome of this isolate using the polymerase chain reaction and then sequenced; data suggest this virus is very closely related to IV 1, originally isolated from Tipula paludosa. These properties indicate this virus is an isolate of the genus Iridovirus.
Chimeric infectious bursal disease virus-like particles expressed in insect cells and purified by immobilized metal affinity chromatography.
Chimeric virus-like particles (VLPs) of infectious bursal disease virus (IBDV) were produced by coinfecting Spodoptera frugiperda (Sf-9) insect cells with two recombinant baculoviruses, vIBD-7 and vEDLH-22. vIBD-7 encodes VP2, VP3, and VP4 of the IBDV structural proteins. vEDLH-22 encodes VP2 with five histidine residues at the carboxy-terminus (VP2H). Coinfection produced hybrid VLPs composed of VP2, VP2H, and VP3. The additional histidine residues on VP2H enabled the efficient purification of VLPs based on immobilized metal affinity chromatography (IMAC). These results demonstrated that the VLPs formed are comprised of chimeric subunits with attached affinity ligands, and further, that sufficient His5 ligand was available for binding to the IMAC metal-chelating resin. Additionally, these novel particles were fully characterized for antigenicity by a series of monoclonal antibodies, and appeared identical to the two wild-type IBDV strains contributing subunits to the chimeric VLP. IMAC purification provides a promising low-cost and simple scheme to purify VLPs as vaccines.
INFECTIONS OF LABORATORY ANIMALS POTENTIALLY DANGEROUS TO MAN: ECTOPARASITES AND OTHER ARTHROPODS, WITH EMPHASIS ON MITES.
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Post-inoculation changes in enzyme activity of Aedes aegypti infected with Chikungunya virus.
Levels of acetylcholinesterase, non-specific esterases, glutathione-S-transferase and glucose-6-phosphate dehydrogenase in Aedes aegypti (L.) mosquitoes inoculated intrathoracally with Chikungunya virus were elevated, as compared to uninoculated control insects. A number of these enzymes are important in the insects defence mechanism against xenobiotics, such as pesticides. Malathion bioassays indicated a reduction in the susceptibility of experimentally injected insects with virus or virus-free inoculum, compared to non-inoculated controls. However, insects which were mock-inoculated (injected with no inoculum) showed a similar reduction in susceptibility suggesting that the observed effect was due to the mobilization of a defence reaction in the mosquitoes in response to injury during inoculation.
UV radiation effects on pathogens and insect pests of greenhouse-grown crops.
Production of high-value crops is often performed under protected cultivation. In recent years various spectral modifications have been made in greenhouse covers. Two of the main reasons to modify the spectral characteristics of greenhouse covers have been to suppress the proliferation of several foliar diseases and to protect crops from insects and insect-borne virus diseases of greenhouse-grown crops. These goals were achieved by complete or partial absorption of solar UV radiation, which interrupts the life cycle of several fungal pathogens and alters the visual behavior of many insects. Examples of these management strategies are described in this article.
Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles.
Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types.
Activation of an insect baculovirus promoter in mammalian cells by adenovirus functions.
The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in insect cell lines in culture. In mammalian cells, however, the virus cannot be propagated. AcNPV DNA does not replicate or persist and is not transcribed in mammalian cells (Tjia et al., 1983). In insect cells productively infected with AcNPV, at least two major late viral gene products have been recognized, the polyhedrin, which makes up the bulk of the polyhedral inclusion bodies in infected cell nuclei, and a 10,000 Da protein (p10) of unknown function. The p10 promoter has been fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as a reporter gene (Knebel et al., 1985). Activity of this construct can be elicited in AcNPV-infected insect cells but not in uninfected insect cells or in mammalian cells. Presumably, the late p10 promoter requires other AcNPV gene products for activity. When the pAcp10-CAT construct is transfected into BHK21 hamster cells at about 18 h after infection with human adenovirus type 5 (Ad5), the insect AcNPV promoter is transactivated in cells of the heterologous mammalian species. The results of S1 protection analyses on the RNA from Ad5-infected and pAcp10-CAT transfected cells reveal that the p10 promoter is used for initiation of transcription. Similarly, the p10 insect virus promoter is activated in BHK21 hamster cells cotransfected with the HindIII-G fragment of adenovirus type 2 (Ad2) DNA which contains the E1A and parts of the E1B region in the left terminal 7.8% of the Ad2 genome. Moreover, in human 293 cells or in BHK297-C131 hamster cells, which both carry and constitutively express the E1 region of Ad5 DNA, the pAcp10-CAT construct is also expressed, and similarly in HE7 hamster cells which carry appreciable portions of the Ad2 genome (Klimkait and Doerfler, 1985). It is concluded that adenovirus functions are capable of transactivating a heterologous insect virus promoter in mammalian cells.
Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells.
The hepatitis E virus (HEV) capsid antigen has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. The full-length HEV ORF2 protein product is predicted to contain 660 amino acids and to weigh 72,000 daltons. Expression of the HEV ORF2 capsid gene from recombinant baculoviruses in insect cells produced multiple immunoreactive proteins ranging in size from 30 to 100 kDa. The most abundant HEV proteins had molecular weights of 72, 63, 56, and 53 kDa. Temporal expression kinetics of these viral polypeptides indicated that the 72- and 63-kDa polypeptides were produced abundantly within the initial 36 h. postinfection but were replaced by 56- and 53-kDa polypeptides in the cell and medium, respectively, by 48 h postinfection. The 53-kDa protein was secreted as early as 24 h. postinfection, and accumulation in the medium peaked by 72 h postinfection. Purification of the 53-, 56-, and 63-kDa viral polypeptides was accomplished by anion-exchange and subsequent gel filtration chromatography. Sequence analysis of the 53-, 56-, and 63-kDa HEV polypeptides indicated that the amino terminus was amino acid residue 112 of the predicted full-length protein product. The results of carboxy terminal amino acid sequencing indicated that the carboxy terminus of the 53-, 56-, and 63-kDa HEV proteins was located at amino acid residues 578, 607, and 660, respectively. The molecular masses of the 53- and 56-kDa HEV polypeptides were 53,872 and 56,144 as determined by mass spectroscopy.