- Volume 66, Issue 6, 1985
Volume 66, Issue 6, 1985
- Review Article
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- Bacterial
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Influence of C-terminal Modifications of øX174 Lysis Gene E on its Lysis-inducing Properties
More LessSUMMARYThe øX174 gene E product (gpE) causes lysis of Escherichia coli by inducing the host autolytic system. Experiments were carried out to ascertain which part of the 91 amino acid polypeptide carries the functional site for this process. For this purpose fusion genes were created comprising the first 51 codons of gene E and unrelated sequences coding for 102 or 33 amino acids respectively. The chimeric protein of 153 amino acids consisting of the N-terminal part of gpE and a fragment of β-galactosidase, was neither able to lyse E. coli nor to restore β-galactosidase activity by α-complementation. Expression of the 84 amino acid polypeptide, however, was able to induce lysis of E. coli. It is therefore concluded that the functional lysis-inducing site of gpE is located within the cloned N-terminal part of gene E. In the shorter chimeric protein the sequence following the functional site was tolerated or necessary for stabilization, but in the longer chimeric protein, the C-terminal sequence disturbed the lysis-inducing conformation.
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Expression of T4 Early Genes 62, 44, 45 and 46 in the λ-T4 Recombinant Phage λ806-17
More LessSUMMARYA λ-T4 recombinant phage, λ806-17, which carries the T4 early genes 62, 44, 45 and 46, was studied inside a homoimmune lysogen. Under such conditions, gene expression from the λ promoters is represented. Results showed extensive expression of gene 46, and significant expression of genes 62 and 45. The expression of these early T4 genes is presumed to depend on T4 promoters included in the cloned fragment. A new promoter proximal to gene 46 is implicated. The results also indicate that the extent of gene expression, in terms of complementation, increases with the time allowed for expression.
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- Animal
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Antigenic Cross-reactivity between Caprine Arthritis-Encephalitis, Visna and Progressive Pneumonia Viruses Involves All Virion-associated Proteins and Glycoproteins
More LessSUMMARYAntigenic relatedness between the virion-associated proteins of caprine arthritis-encephalitis, visna and progressive pneumonia viruses was examined. Antigenic cross-reactivity was assessed by immunoprecipitation of disrupted, radiolabelled virus with goat, sheep and rabbit antisera, followed by resolution of the immunoprecipitation products by SDS-polyacrylamide gel electrophoresis. The results indicate that antigenic cross-reactivity between the caprine and ovine virus isolates involves all of the major virion-associated proteins and glycoproteins. The common antigenic determinants exhibited by virion structural proteins are immunogenic in goats, sheep and rabbits.
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Respiratory Syncytial Virus Polypeptides. V. The Kinetics of Glycoprotein Synthesis
More LessSUMMARYThe cell-associated glycoproteins of respiratory synctial virus included GP1 (90K), VP70 (70K), VGP48 (48K) and GP26 (26K). The time course of virus protein synthesis in HeLa cells revealed that the nucleocapsid protein (VPN41) was the first to appear at 11 h post-infection followed by the appearance of the other viral proteins at 16 h post-infection. Pulse-chase experiments with [3H]leucine or [35S]methionine demonstrated that the precursor for VGP48 and GP26 was an uncleaved protein of 70K (VP70) which required at least 30 min to chase into its final products, while the precursor for GP1 was a glycoprotein of 46K, and also required at least 30 min to chase into GP1. Trypsin treatment of monensin-treated infected cells suggested that VP70 can be cleaved intracellularly into its products VGP48 and GP26.
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Comparative Pathology of Isolates of Spodoptera frugiperda Nuclear Polyhedrosis Virus in S. frugiperda and S. exigua
More LessSUMMARYSpodoptera frugiperda nuclear polyhedrosis virus was highly pathogenic to both S. frugiperda and S. exigua. Plaque-purified variants from two of the original isolates showed much greater differences in pathogenicity to the two insect species than the original isolates. Plaque-purified variants from one of the isolates (D) nearly lost pathogenicity for S. exigua while remaining pathogenic for S. frugiperda. Some of the plaque-purified variants produced atypical symptoms, even in S. frugiperda. These variants did not liquefy larvae and release polyhedra when the larvae died as is typical for nuclear polyhedrosis infections in Lepidoptera. These variants also produced fewer polyhedra per g larval weight and often had fewer virions per polyhedron. Light and electron microscopical studies of S. frugiperda and S. exigua infected with one of the original isolates (A) and two of the plaque-purified variants (B2 and D7) indicated that only S. frugiperda infected with isolate A had the highly productive infection and viral morphogenesis typical of nuclear polyhedrosis viruses. Exposure of S. frugiperda to isolate B2 or D7 resulted in a slightly delayed infection characterized by increased amounts of abnormal viral morphogenesis and polyhedra of decreased size. S. exigua infected with isolate A or B2 had greatly reduced and delayed infections that were accompanied by highly variable abnormal viral morphogenesis; virtually no normal polyhedra were produced in these instances. Isolate D7 produced neither nucleocapsids nor polyhedra in S. exigua. Large paracrystalline aggregates of nucleocapsids were common in S. frugiperda infected with isolates B2 or D7 and in S. exigua infected by isolate A. Infection of S. exigua by isolate A or B2 was typified by the accumulation of large amounts of excess envelope membrane in the form of strands and vesicles of various sizes. Normal virogenic stromata were characteristics only of S. frugiperda infected with isolate A or B2. Polyhedra produced in S. exigua by even the most pathogenic isolates (A and B2) contained few if any normal virions and were not infective for either S. frugiperda or S. exigua.
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The Nucleotide Sequence of the Pieris brassicae Granulosis Virus Granulin Gene
More LessSUMMARYTwo overlapping restriction fragments containing the Pieris brassicae granulosis virus (GV) granulin gene were cloned into plasmids. The regions containing the coding region and the 5′ and 3′ flanking regions were subcloned into M13 and sequenced. The nucleotide sequence data were compared to those for the granulin gene from the Trichoplusia ni GV and the polyhedrin gene from the Autographa californica nuclear polyhedrosis virus (AcMNPV). The amino acid sequences derived from these DNA sequences indicated that the two GV proteins are more closely related to each other (77% amino acid homology) than either is to the AcMNPV (about 53% amino acid homology for either GV). The N-terminal region shows the greatest degree of variation between these proteins. Highly conserved amino acid sequences were identified between the two GVs and were also found between NPVs. Certain of these conserved regions are shared between GVs and NPVs while others are not.
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Isolation of Haemorrhagic Fever with Renal Syndrome Virus from Leukocytes of Rats and Virus Replication in Cultures of Rat and Human Macrophages
SUMMARYNewborn rats were inoculated intraperitoneally with haemorrhagic fever with renal syndrome (HFRS)-related virus (B-1 strain), and virus isolation from their various organs was attempted between 1 and 25 weeks after inoculation. Virus could be isolated repeatedly from lung, brain, spleen and kidney and also from peripheral blood. When virus isolation was carried out on fractionated peripheral blood cells, virus was associated mainly with the macrophage fraction and to a lesser extent with granulocytes. Virus replicated well in peritoneal exudate cells of normal rats and it grew in the adherent mononuclear cells from normal human peripheral blood. These data suggest that macrophages, permissive for HFRS-related virus replication, might contribute to the spread of viral infection in vivo.
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Two Electropherotypes of Bluetongue Virus Serotype 2 from Naturally Infected Calves
More LessSUMMARYThe first isolation of bluetongue virus (BTV) serotype 2 in the U.S.A. was in 1982 from a sentinel herd of cattle at Ona, Florida. Electrophoretic analysis of genome RNA revealed that all 16 serotype 2 isolates obtained from this focus of infection had one of two electropherotypes (designated Ona A and Ona B). All genome segments of Ona A and Ona B had detectable differences in electrophoretic mobility, with major differences noted for segments 1, 4, 5, 7, 8, 9 and 10. Electrophoretic comparison revealed that Ona A was indistinguishable from the African serotype 2 prototype isolated 23 years earlier. In 1983, Ona B, in the apparent absence of Ona A, was isolated from two additional cattle herds in Florida. These results suggest that Ona B may be a variant of Ona A that evolved as a result of mutation or reassortment with another BTV strain, and may be better adapted to the selective pressures found in southern Florida. Comparison of the electropherotypes of Ona B with two Florida isolates of serotype 13 and 17 and the prototypes of BTV 10, 11, 13 or 17 produced no evidence for reassortment between Ona A and any of these strains as the possible origin of Ona B.
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The Nucleocapsid of Berne Virus
More LessSUMMARYIn Berne virus-infected cells and in gradient-purified virions two major proteins with mol. wt. of 20K and 22K were detected. The 22K species is thought to represent the main envelope polypeptide; in infectious culture media it was present in a low density substructure which could be quantitatively converted into slowly sedimenting material by detergent treatment. The 20K polypeptide (accounting for about 80% of the 14C-amino acid label in the virion) was phosphorylated, occurred in an intracellular substructure of higher density than the virion (ρ = 1.36 g/ml in CsCl) and was the only viral protein possessing RNA-binding properties; it was recognized preferentially by heterologous animal sera in immune precipitation. The 20K species is therefore identified as the main capsid protein. Two additional polypeptides (19K and 17K) were regularly detected in extracts of infected cells; they appeared to share oligopeptides with the 20K protein and are interpreted as being proteolytic cleavage products. The nucleocapsid of Berne virus was visualized after ether treatment as a flexible bacilliform structure with conspicuous transverse striation. Demonstration of a 20K nucleocapsid protein further supports the authors′ proposal that Berne virus is a representative of a new family of enveloped RNA viruses (Toroviridae).
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Protection from Herpes Simplex Virus-induced Neuropathology in Mice Showing Delayed Hypersensitivity Tolerance
More LessSUMMARYHerpes simplex virus (HSV)-susceptible mice inoculated under conditions favouring the preferential activation of T suppressor (Ts) cells acting on the delayed-type hypersensitivity (DTH) response to the virus were protected from lethal herpes encephalitis and from central nervous system (CNS) demyelination (as reflected by ear paralysis), compared to controls given normal priming. Thus, suppressed DTH was not incompatible with recovery from acute infection and may indeed have been beneficial. Protection could be transferred by T cells from donors given a ‘DTH-tolerogenic’ priming regime. It was unlikely that protection resulted from enhancement of other mechanisms such as cytotoxic T cell activation, antibody or interferon production, since no reduction of virus spread was observed in protected mice. In addition, several aspects of Ts cell activation by intravenous inoculation of avirulent HSV type 1 have been characterized. Suppression was virus dose-dependent and could be transferred to the efferent limb of a DTH response. Activation of Ts cells for DTH coincided with an enhanced antibody response. It is suggested that protection in this model may be mediated by Ts cells which act to limit DTH-mediated immunopathology in the CNS.
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Isolation and Characterization of Deletion Mutants of Herpes Simplex Virus Type 2 (Strain HG52)
More LessSUMMARYWe provide evidence that: (i) two variants lacking the XbaI site at map coordinate 0.7 have been selected following XbaI treatment of the DNA of herpes simplex virus type 2 strain HG52; (ii) one of these mutants had lost the 0.7 restriction site due to a deletion of approximately 150 base pairs and in the other the site loss was due to a similar sized sequence insertion; (iii) following XbaI treatment, four variants with deletions ranging in size from 1.5 kb (in both TRL and IRL) to 9 kb in IRL were isolated; (iv) substantial deletions in the long terminal repeat regions of HG52 are present with a frequency of 24% of genomes in the elite stock, a variant with a 3.75 kb deletion in IRL making up 10% and one with a 1.5 kb deletion in both IRL and TRL making up 14%; (v) two of the variants isolated after XbaI treatment of viral DNA were identical to the deletion prototype within the elite stock, suggesting that these variants were not generated as a result of XbaI treatment but pre-existed in the viral DNA pool; (vi) the deletion variants were stably maintained during routine stock propagation, were viable and could be propagated as cloned populations; (vii) the deletions did not have a marked deleterious effect on the one-step growth kinetics of the virus.
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Genetic Relationships of the Dengue Virus Serotypes
More LessSUMMARYPrevious studies have compared RNA genomes from the different dengue virus serotypes by cDNA-RNA hybridization using dengue-1 virus- and dengue-2 virus-specific cDNA probes. These probes revealed that there is a close genetic relationship between dengue virus serotypes 1 and 4. In this communication, the cDNA-RNA hybridization results using dengue-3- and dengue-4-specific cDNA probes to determine the genetic relatedness of all four dengue virus serotypes are reported. The results indicate that serotypes 1 and 4 are genetically very closely related (sharing about 70% of their genomes as detected by both the dengue-1 and dengue-4 cDNA probes), as are serotypes 3 and 4 (sharing about 50% of their genomes as detected by both the dengue-3 and dengue-4 cDNA probes). Serotype 2 does not seem to be very closely related to the other dengue virus serotypes by cDNA-RNA hybridization analysis.
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Induction of Influenza A Virus Cross-reactive Cytotoxic T Cells by a Nucleoprotein/Haemagglutinin Preparation
More LessSUMMARYAn ammonium deoxycholate fraction from bromelain-treated influenza A virus was highly enriched for virus nucleoprotein and contained residual haemagglutinin (NP/HA). The preparation did not contain detectable levels of matrix or neuraminidase proteins and was free of infectious virus. NP/HA effectively primed mice for cytotoxic T cells which lysed syngeneic cells infected with any type A influenza virus. Furthermore, NP/HA generated A-type virus cross-reactive cytotoxic T cells when added in vitro to spleen cells from mice previously primed with infectious influenza A virus. These properties imply that NP/HA has potential as a vaccine for heterotypic influenza A immunity.
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Cytotoxic T Cell Lysis of Target Cells Fused with Liposomes Containing Influenza Virus Haemagglutinin and Neuraminidase
More LessSUMMARYThe lytic activity of secondary cytotoxic lymphocytes against influenza A virus was tested on cells which had been fused with liposomes containing the haemagglutinin and the neuraminidase of an avian influenza A virus (fowl plague virus, FPV). Fusion was obtained solely by the activity of the haemagglutinin and neuraminidase incorporated into the liposomes, without the need for any additional fusion factor. Highly reproducible lysis of these FPV-liposome target cells by influenza A-specific cytotoxic cells was found. In contrast, target cells containing the glycoproteins HN and F of Newcastle disease virus (NDV) were not lysed. In almost all experiments effector cell populations capable of lysing target cells also lysed the natural killer cell (NK)-sensitive cell line YAC-1. However, high NK activity alone was not sufficient to lyse target cells fused with liposomes containing the viral surface glycoproteins. To our knowledge this is the first report where after artificial introduction of viral surface components into cell membranes (either by fusion or by transfection) lysis of target cells was monitored also for non-specific lysis mediated by NK-like cells. Both the H-2 restriction and the virus specificity of lysis of FPV-liposome target cells indicate that influenza virus haemagglutinin and possibly neuraminidase do function as target antigens for influenza-specific T cells.
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Enzyme Immunoassay of Interferon with Peroxidase-labelled Virus-specific Monoclonal Antibodies
SUMMARYTo quantify the antiviral effect of interferon (IFN) we applied a mixture of two horseradish peroxidase-labelled monoclonal antibodies, specific for the El glycoprotein of Semliki Forest virus, in a direct enzyme immunoassay. This assay is suitable for detection of virus replication in L-cells, seeded as monolayers in 96-well plates. Inhibition of absorbance values caused by IFN was determined in a Flow Titertek Multiskan. Three IFN samples from different sources were titrated simultaneously in the enzyme immunoassay and in the vesicular stomatitis virus plaque reduction test in five consecutive experiments. Titres were calculated as the inverse value of the dilution of IFN causing 25% inhibition of absorbance values and 50% reduction of plaque counts respectively. The results show equality of precision and reproducibility between and within the two assays. However, the enzyme immunoassay is more convenient and objective than the plaque reduction assay.
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- Plant
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Infection of Protoplasts Derived from Potato Shoot Cultures with Potato Virus X
More LessSUMMARYBecause it contains the Nx gene, potato cv. ‘Maris Piper’ reacts hypersensitively to inoculation with group three strains of potato virus X (PVX). However, protoplasts prepared from aseptically grown shoot cultures of potato cv. ‘Maris Piper’ were infected reproducibly with either PVX or PVX RNA using inocula containing polyethylene glycol. The extent of infection of protoplasts was assessed by fluorescent antibody staining, and by assaying extracts for antigen by ELISA, for infectivity and for PVX RNA content by cDNA hybridization. Infection occurred in 43 to 65% of the protoplasts infected with PVX and in 57 to 70% of the protoplasts infected with PVX RNA. In comparison, when high quality protoplasts were obtained from leaves of pot-grown plants 55 to 65% became infected when inoculated with PVX RNA. However, protoplasts obtained from leaves of whole plants varied greatly in both quantity and quality, whereas shoot cultures reproducibly gave high quality protoplast preparations in large numbers. In protoplasts from either source, yields of progeny virus were between 10 and 30 pg per infected protoplast. The multiplication of a group 3 strain of PVX in protoplasts containing the Nx gene did not induce necrosis and confirms that hypersensitivity is not always expressed in isolated protoplasts.
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Translation Products of RNA from Indian Peanut Clump Virus
More LessSUMMARYWhen RNA from particles of Indian peanut clump virus (IPCV) was translated using messenger-dependent reticulocyte lysate the largest product had a mol. wt. of about 195000 and the other main products had mol. wt. of 143000 and 24500. The 24500 mol. wt. polypeptide co-migrated with IPCV coat protein and was the only product that reacted with antiserum to IPCV particles. Translation of separated RNA species showed that the 195000 mol. wt. and 143000 mol. wt., as well as some smaller less abundant products, arose from translation of RNA-1 (mol. wt. 1.9 × 106) whereas RNA-2 (mol. wt. 1.6 × 106) was translated to give only the 24500 mol. wt. product Translation of RNA of mol. wt. 0.5 × 106 to 0.9 × 106 gave a 50000 mol. wt. product that was present and sometimes prominent in translation products of unfractionated RNA. A product of about 20000 mol. wt. was made by translation of RNA of about 0.2 × 106 mol. wt. IPCV resembles the viruses with bipartite genomes whose larger RNA species is translated into large polypeptides and whose coat protein gene is on the smaller RNA species. The other translation products may be those of subgenomic messenger, or of satellite, RNA species.
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- Fungal
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Isolation of a Virus from Virulent Strains of Rhizoctonia solani
More LessSUMMARYDouble-stranded RNA viruses were detected in virulent strains of Rhizoctonia solani. The virus particles were 33 nm in diameter, contained two or three major segments of dsRNA (mol. wt. 1.6 × 106, 1.45 × 106 and 1.25 × 106), had a density of 1.34 g/ml and had an estimated sedimentation coefficient of 161S. The major coat protein had a mol. wt. of 46000. An RNA-dependent RNA polymerase activity was associated with the viral capsids. Native hypovirulent strains of R. solani were devoid of these viruses and attenuation of virulent strains to hypovirulence appeared to be correlated with the loss of some or all of the segments of dsRNA. Transmission of dsRNA from a virulent to a hypovirulent strain by heterokaryon formation was associated with the transmission of virulence.
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