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Volume 57,
Issue 2,
1981
Volume 57, Issue 2, 1981
- Bacterial
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Comparison of the Lipid-containing Bacteriophages PRD1, PR3, PR4, PR5 and L17
More LessSUMMARYFive broad host range lipid-containing bacteriophages PRD1, PR3, PR4, PR5 and L17 isolated from different parts of the world were compared using biological and structural criteria. Virus morphology as well as genome sizes appeared to be identical. However, these viruses could be distinguished by restriction endonuclease mapping and by their structural protein patterns in SDS-gel electrophoresis. The viruses studied thus form a very close group of lipid-containing bacteriophages. We suggest PRD1 as a model organism for this group and that the group be called the PRD1 phage group.
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- Animal
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Comparison of Bluetongue Type 20 with Certain Viruses of the Bluetongue and Eubenangee Serological Groups of Orbiviruses
More LessSUMMARYThe genome of bluetongue virus type 20 consists of 10 segments of double-stranded RNA each of which contains unique sequences as determined by oligonucleotide mapping. The 10 polypeptide products of the virus genome were detected in virus-infected cells, and in pulse-chase experiments there was no secondary cleavage of the primary gene products. Using stringent conditions for RNA-RNA reassociation, no significant homology could be detected between the genomes of bluetongue type 20 isolated in Australia and representative serotypes isolated in other geographic regions. The results suggest sequence divergence between geographically isolated viruses and not the recent introduction of a bluetongue virus into Australia.
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Homogeneity of the Structural Glycoprotein from European Isolates of Tick-borne Encephalitis Virus: Comparison with Other Flaviviruses
More LessSUMMARYIsolates of tick-borne encephalitis (TBE) virus from Finland, Germany, Czechoslovakia, Switzerland and Austria were compared with strains of the Far Eastern subtype isolated in Russia as well as Louping ill virus and other flaviviruses belonging to a different serocomplex: West Nile, Murray Valley encephalitis and Rocio viruses. Analysis of the structural polypeptides by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed identical mol. wt. of the glycoprotein E (mol. wt. 55000) and the core protein C (mol. wt. 15000) for all the TBE virus strains analysed. However, the small envelope protein M from viruses isolated in Germany, Switzerland and Austria migrated slightly slower (apparent mol. wt. 7500) compared to M from viruses isolated in Finland, Czechoslovakia or the Far Eastern subtype strains (apparent mol. wt. 6500 to 7000). The structural glycoproteins were isolated from purified [35S]methionine-labelled virions and subjected to peptide mapping by limited proteolysis with α-chymotrypsin or V8 protease followed by SDS-PAGE of the resulting cleavage products. With both proteases a remarkably homogeneous pattern was obtained for all the European isolates with only very minor deviations from the common pattern in single cases. Similar but distinguishable patterns were obtained for the Far Eastern subtype strains and also Louping ill virus, which, in addition, differed in the mol. wt. of its core protein C (mol. wt. 16000) and the small membrane protein M (mol. wt. 9000). These almost identical peptide maps observed with the TBE virus strains were in sharp contrast to the unrelated patterns obtained with the glycoproteins from West Nile, Murray Valley encephalitis and Rocio viruses. Although these viruses are serologically closely related and members of the same serocomplex of flaviviruses their glycoprotein peptide maps were completely different from one another. In a competitive radioimmunoassay all European TBE virus isolates showed identical immunological reactivity which further points to the great stability of this type of virus.
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Interferon Induction by Lymphocytic Choriomeningitis Viruses Correlates with Maximum Virulence
More LessSUMMARYLymphocytic choriomeningitis (LCM) viruses isolated from the blood of persistently infected mice, could be clearly divided into two categories by observing the disease patterns they produced after intracerebral (i.c.) injection in a number of adult inbred and outbred mice. One type (aggressive) caused the classic pattern of convulsive death in 100% of the mice 7 to 9 days after infection, while the other (docile) caused a protracted disease with deaths occurring, if at all, 2 to 4 weeks after infection. Interferon could be detected in the serum of adult mice on the 3rd and 4th day after infection with several independently cloned aggressive, but not docile, viruses. The inability of docile virus to induce interferon was not due to poor or delayed virus replication in the brain. The aggressive pattern of disease could be provoked easily in docile virus-infected mice with the interferon inducers poly(rI).poly(rC), tilorone hydrochloride or Newcastle disease virus. The amount of interferon produced had little effect on the mean day of death. Mice that differed over 10-fold in their serum interferon levels after LCM infection, either by genetic predisposition or by stimulation with increasing amounts of poly(rI).poly(rC), presented almost identical patterns of mortality.
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Persistence and Expression of Marek’s Disease Virus DNA in Tumour Cells and Peripheral Nerves Studied by in situ Hybridization
SUMMARYWe have used cloned fragments of Marek’s disease virus (MDV) DNA and in situ hybridization to search for virus DNA and study its expression in infected chick embryo fibroblasts (CEF), lymphoblastoid cell lines, tumours and neural lesions. DNA from the HPRS 16/att strain of MDV was cleaved with EcoRI endonuclease and several fragments were cloned in Escherichia coli using the vector PBR322. Seven fragments ranging in size from 2.6 to 11 kbp representing approx. 25% of the MDV genome were labelled in vitro and annealed to EcoRI digests of DNA from infected cells and tumours following separation and transfer according to the Southern blotting procedure. Most of the selected MDV DNA fragments hybridized to fragments of corresponding sizes in EcoRI digests of DNA from cell lines and tumours and failed to hybridize to digests of uninfected chick cell DNA. In situ hybridization using 3H-labelled DNA with specific activity of 108 d/min/µg as probe showed intranuclear MDV DNA in infected CEF, in every cell of two lymphoblastoid cell lines and in the majority of infiltrating or proliferating lymphoid cells found in type ‘A’ lesions of grossly enlarged peripheral nerves. Both intranuclear and cytoplasmic RNA were detected in cells that contained virus DNA. However, comparatively little virus RNA appears to be transcribed in cell lines and in infected tissues from the regions of virus DNA (25% of genome) used as probe in this study. Our results favour the hypothesis that the accumulation of lymphoid cells in nerves is not the result of an inflammatory response to infected nerve cells but is rather the consequence of proliferating transformed cells.
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Complementation between Phosphonoacetic Acid-resistant and -sensitive Variants of Herpes Simplex Viruses: Evidence for an Oligomeric Protein with Restricted Intracellular Diffusion as the Determinant of Resistance and Sensitivity
More LessSUMMARYComplementation between phosphonoacetic acid (PAA)-resistant (P r) and -sensitive (P s) variants of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) was studied to provide information on the function of the virus-coded DNA polymerase. Complementation within and between serotypes was demonstrated, with the growth of the P s partner in mixed infections becoming relatively more resistant and the P r partner relatively more sensitive to PAA than in the corresponding single infections. However, the relative contribution of the P s partner to the mixed infection had a disproportionately large effect on the resultant sensitivity of the mixed infection which was incompatible with non-interactive (e.g. monomeric) polymerase molecules as determinants of PAA sensitivity and resistance. Although a number of solutions gave equally good fits to the available data, the simplest was obtained by assuming that the functional DNA polymerase was a trimer and that only the (P r)3 homotrimer was active in the presence of the drug. In addition, yields from mixed infections in the presence of PAA were enriched for the resistant partner relative to yields in the absence of the drug. These latter results suggested that the intracellular distribution of resistant DNA polymerase oligomers was non-random with respect to resistant and sensitive template genomes and that these resistant polymerase molecules were more likely to encounter and replicate resistant than sensitive genomes. Such an explanation seems to require vectorial nuclear-cytoplasm-nucleus translocation and restricted diffusion of transcript and gene products determining resistance.
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Analysis of the Genome of Equine Herpesvirus Type 1: Arrangement of Cleavage Sites for Restriction Endonucleases EcoRI, BglII and BamHI
More LessSUMMARYThe genome of an Australian isolate of equine herpesvirus type 1 (equine abortion virus) has been analysed using the restriction endonucleases EcoRI, BglII and BamHI, and a physical map constructed. Terminal fragments were identified by exonuclease treatments, and linkage of fragments was deduced by a combination of single- and double-digest experiments and cross-blot hybridizations. The genome has a mol. wt. of 100 × 106 and is comprised of a short unique region bounded by repetitive sequences, which is present in both orientations in approximately equal amounts in the DNA population, and a long unique region existing in only one orientation.
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Demonstration of the Immunogenicity of Hepatitis B Core Antigen in a Hepatitis B e Antigen Polypeptide (P19)
More LessSUMMARYThe core of Dane particles, the presently accepted hepatitis B virus nucleocapsid, contains two polypeptides (P19 and P45) with the antigenicity of hepatitis B e antigen (HBeAg). The antigenicity of hepatitis B core antigen (HBcAg) was not detectable in either of them by the conventional in vitro assay methods, despite the fact that both of these polypeptides were derived from the core of Dane particles. When a rabbit had been immunized with the purified preparation of P19 emulsified in complete Freund’s adjuvant, however, humoral antibody against HBcAg was produced in addition to the antibody against HBeAg. Amino acid analysis of P19 disclosed a high content of arginine (12.9%), leucine (11.9%), serine (10.3%) and proline (10.2%). The amino acid composition of P19 was found to be strikingly similar to the composition of the 183 amino acid sequence deduced from the sequence of hepatitis B virus DNA which has been presumed to be encoding HBcAg. We conclude that both HBeAg and HBcAg are antigenic determinants borne by the major polypeptide (P19) constituting the core of Dane particles.
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The Physicochemical Properties of Infectious Hepatitis A Virions
More LessSUMMARYThe propagation of hepatitis A virus (HAV) in the cell line PLC/PRF/5 made possible the radiolabelling in vivo of mature, infectious hepatitis A virions and the determination of their physicochemical properties. In contrast to poliovirus type 2 (160S, 1.340 g/ml), HAV had a sedimentation coefficient of 156 ± 2S and a buoyant density of 1.332 g/ml in CsCl. The genome of HAV consisted of linear single-stranded RNA which sedimented at 32.5S under non-denaturing conditions. Compared to the size and sedimentation behaviour of poliovirus RNA (2.6 × 106 mol. wt., 35S) this corresponds to a mol. wt. of 2.3 × 106. Electrophoresis under fully denaturing conditions, however, revealed a mol. wt. of 2.8 × 106 and indicates the existence of relatively extended regions with secondary structure. The purified virus genome, containing a poly(A) sequence, served as a messenger for the synthesis of virus antigen in PLC/PRF/5 cells. Finally, in accordance with previous observations, the capsid of the virion was found to be constructed of three major polypeptides (VP1, 31 × 103; VP2, 26 × 103; VP3, 21 × 103 mol. wt.) and of two less readily demonstrable components probably corresponding to VP4 (8 × 103 to 10 × 103 mol. wt.) and the precursor polypeptide VP0 (40 × 103 mol. wt.).
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Restricted HEL-12 Virus Infection in de novo Infected Human and Canine Cells
More LessSUMMARYModel systems to study restricted primate retrovirus expression were established by de novo infection of canine foetal thymus cells (CF-2Th) and superinfection of HEL-12 cells with HEL-12 virus. In the resulting CF-2Th/HEL-12V cells and HEL-12/HEL-12V cells, four sequential stages of virus infection were defined by the production of reverse transcriptase (RT)-containing particles and expression of virus antigens as detected by radioimmunoassays. Stage 1 cells did not synthesize virus antigens or produce RT-containing particles. Stage 2 cells synthesized virus antigen but not RT-containing particles. Stage 3 cells synthesized antigen and produced RT-containing particles, and stage 4 cells synthesized virus antigens but no longer produced RT-containing particles. The duration of the four stage infection is 2 to 3 weeks in both cell types. Monospecific competition radioimmunoassays to detect HEL-12V p30 or gp70 antigen showed high levels of virus antigen throughout stages 2 to 4 of infection. Analysis of immunoprecipitates formed under conditions to detect either p30- or gp70-containing proteins in cells pulsed and pulsed-chased with [3H]eucine showed the same spectrum of virus precursor polyproteins, intermediates and mature virion components in stage 2 to 4 cells in canine and human infections. Spent culture fluids collected from stage 3 and stage 4 CF-2Th/HEL-12V cells failed to reveal inhibitors of RT activity. Stage 4 CF-2Th/HEL-12V or HEL-12/HEL-12V cells labelled with [3H]uridine produced virions which incorporated [3H]uridine but did not have RT activity, suggesting that restricted infection is characterized by production of HEL-12V defective in RT activity.
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Antigenic Characterization of Measles and SSPE Virus Haemagglutinin by Monoclonal Antibodies
More LessSUMMARYHybrid cells secreting monoclonal antibodies directed against the haemagglutinin (H) protein of measles virus (Edmonston) were produced by fusion of mouse myeloma cells with spleen cells derived from immunized mice. Measles antibodies secreted by these cells were tested for their ability to react with measles virus in immunoprecipitation experiments and assays of binding, neutralization, haemagglutination inhibition and haemolysin inhibition. On this basis 21 out of 75 hybridomas could be defined and divided into five functional groups with different properties. However, when tested against other measles virus strains, including those isolated from subacute sclerosing panencephalitis (SSPE) patients, normalized radioimmunoassay (RIA) binding titres showed that the extent to which a given antibody bound could vary greatly with the virus strain examined. Moreover, the biological actions within a group were found to be very heterogeneous, even when high antibody binding titres were observed. These results suggest that different measles virus strains, which are not distinguishable by polyvalent sera, do in fact possess antigenic differences. Furthermore, the functional significance of a given virus epitope may vary from strain to strain. Hybridoma antibodies were also used to demonstrate the occurrence of antigenic changes within the H polypeptide of SSPE virus during the course of a non-productive, persistent infection in vitro.
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Detection of Antigenic Variation of Influenza A Matrix Protein by a Competitive Radioimmunoassay
More LessSUMMARYAntigenic relationships among the matrix proteins of influenza A viruses were analysed using a competitive radioimmunoassay technique. There were at least two antigenic determinants on the M protein of A/PR/8/34 virus. One antigenic determinant was shared by several influenza A viruses of human, avian, swine or equine origin, while the second determinant was not necessarily shared by all influenza viruses since it was not detected on the M protein of A/chicken/Germany/N/49 virus. In addition, the cross-reactive determinant(s) of the M protein of N virus was not equally represented in other influenza A virus subtypes.
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Evidence for the Involvement of Influenza A (Fowl Plague Rostock) Virus Protein P2 in ApG and mRNA Primed in vitro RNA Synthesis
More LessSUMMARYEleven temperature-sensitive (ts) mutants of influenza A (fowl plague, Rostock) virus were analysed for in vitro RNA transcriptase activity in reactions primed by ApG or globin mRNA at 31 °C or at 40.5 °C, the restrictive temperature for ts mutant growth. Only those ts mutants studied which were defective in RNA segment 1, coding for the virion P2 protein, were defective in RNA transcriptase activity when compared to wild-type virus. Mutants having a defect in the P2 protein had no significant RNA transcriptase activity in reactions at 40.5 °C primed by globin mRNA. However, one mutant showed RNA transcriptase activity similar to wild-type virus at 40.5 °C when ApG (0.3 mm) was used as primer. The results suggest that influenza (fowl plague, Rostock) P2 protein is directly involved in the mRNA priming reaction, as well as in the RNA transcription reaction in vitro.
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Murine Cytomegalovirus Particle Types in Relation to Sources of Virus and Pathogenicity
More LessSUMMARYMurine cytomegalovirus (MCMV) preparations from mouse embryo fibroblasts and from infected salivary glands were purified on potassium tartrate density gradients and examined by electron microscopy. The cell culture virus contained multicapsid enveloped virions which are ruptured easily and account for the excess number of free capsids in these preparations. Salivary gland virus consisted of single-capsid enveloped virus and equal numbers of free capsids. Particle types were imperfectly separated on density gradients, but successful separation was achieved by filtration through 220 and 450 nm Millipore membrane filters. Naked capsids were not infectious and could not be rendered infectious by centrifugal adsorption, showing that centrifugal enhancement of MCMV infectivity is not mediated through this mechanism. Although present in excess number in (avirulent) cell culture virus preparations, naked capsids did not interfere with the action of (virulent) salivary gland virus in newborn mice.
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Serial Propagation of Astrovirus in Tissue Culture with the Aid of Trypsin
T. W. Lee and J. B. KurtzSUMMARYAstrovirus could be serially passed at least 13 times in primary human embryo kidney (HEK) cells when 10 µg/ml of crystalline trypsin was incorporated in a serum-free maintenance medium. In the presence of trypsin the virus was also passed and adapted to a continuous line of rhesus monkey kidney cells (LLCMK2) and primary baboon kidney (PBK) cells in which it was passed 25 and 16 times respectively, without evidence of diminishing infectivity. Attempts to adapt the virus to other cell lines (Vero, Hep II, MRC-5, BHK and HRT-18) were unsuccessful. After 11 passages in HEK cells, a titration of virus grown in different concentrations of trypsin showed that virus propagation was still trypsin-dependent.
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- Plant
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A Satellite Double-stranded RNA in a Virus from Gaeumannomyces graminis
More LessSUMMARYThe double-stranded (ds)RNA components of two serologically related viruses (019/6-A and 38-4-A), obtained from Gaeumannomyces graminis, have been compared. 019/6-A virus has two RNA components, ds1 and ds2, whereas 38-4-A virus has three RNA components, ds1, ds2 and ds3. T1 oligonucleotide fingerprint analysis and saturation hybridization experiments indicated that 019/6-A ds1 and 38-4-A ds1 were very similar, whereas 019/6-A ds2 and 38-4-A ds2 were about 50% homologous. 38-4-A ds3 had little homology with ds1 or ds2 of either virus. The single-stranded (ss)RNA transcripts of 38-4-A virus ds1, ds2 and ds3 each directed the synthesis in vitro of a single polypeptide, with mol. wt. 62000, 55000 and 52000 respectively. Peptide mapping showed that these three polypeptides were unrelated in sequence and that ds2 encoded the virus capsid polypeptide. The ds3 is probably not required for the replication of 38-4-A virus and may be regarded as a satellite RNA.
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Infection of Soybean Protoplasts from Cell Suspension Culture with Bean Pod Mottle Virus
More LessSUMMARYProtoplasts were isolated enzymically from soybean (Glycine max L. ‘Harosoy 63’) callus continuously maintained as suspension cultures. The protoplasts were inoculated with bean pod mottle virus (BPMV) using a medium consisting of 0.4 m-sorbitol, virus, poly-l-ornithine (PLO), buffer and inorganic salts. A concentration of 0.5 × 105 to 1.0 × 105 protoplasts per ml was optimum for efficient virus infection. Although PLO was not essential for infection, it was stimulatory. A combination of PLO and CaCl2 had what appeared to be a synergistic effect in enhancing infection, especially at low virus concentrations. When virus was preincubated for 15 min with CaCl2 or MgCl2 prior to inoculation, both at 0.5 mm or with potassium phosphate buffer at 10 mm pH 5.6, infection of protoplasts was significantly increased over virus which had not been preincubated. The infection process was independent of temperature under the conditions tested. BPMV and the previously introduced cowpea mosaic virus, both comoviruses, showed some contrasting differences in requirements for PLO, pH optimum and temperature.
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Limited Infection of Cereal Leaf Protoplasts by Barley Yellow Dwarf Virus
More LessSUMMARYOat and barley leaf protoplasts that were readily infected with brome mosaic virus could only be infected with barley yellow dwarf virus (BYDV) at low levels of efficiency. Of the conditions tested for infecting the protoplasts with BYDV, the most reliable combination was 2 µg/ml of virus and 0.2 µg/ml poly-l-ornithine with 10 mm-citrate buffer at pH 5. Tobacco protoplasts could not be infected with BYDV under these conditions.
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- Corrigendum
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