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Volume 48,
Issue 1,
1980
Volume 48, Issue 1, 1980
- Review Article
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- Animal
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Prevention of Vaccinia Lesions in Rhesus Monkeys by Human Leucocyte and Fibroblast Interferon
More LessSUMMARYThe prophylactic antiviral activity of systemically administered human interferon preparations was tested in 36 rhesus monkeys against vaccinia virus injected into the skin. All nine control monkeys developed typical vaccinia skin lesions. Eight of nine monkeys treated with daily intramuscular injections of leucocyte interferon (5 × 105 units/kg) from day -1 to day +7 after vaccination were completely protected. No lesions developed after discontinuation of therapy, Administration of the same amounts of leucocyte interferon intravenously (i.v.) was equally effective. Daily intramuscular (i.m.) injections of lower doses of leucocyte interferon (1.25 × 105 units/kg; 0.5 × 105 units/kg) decreased the severity of the skin lesions. Lesion scores correlated inversely with the dose of interferon. Four of six animals receiving daily i.m. injections of fibroblast interferon (5 × 105 units/kg) and one of three animals treated i.v. with the same dose were protected against vaccinia virus, and the lesions in the other monkeys were smaller. Intramuscular injections of 5 × 105 units/kg of fibroblast interferon or 1.25 × 105 units/kg of leucocyte interferon resulted in comparable serum levels and had comparable efficacy in reducing lesion scores.
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Glycopeptide Composition of Hepatitis B Surface Antigen
More LessSUMMARYThree major polypeptides of hepatitis B surface antigen (HBsAg), with mol. wt. 22000 (p22), 27000 (p27) and 68000 (p68), were separated by preparative SDS-PAGE. These three peptides as well as intact HBsAg were found to have almost identical amino acid compositions and carbohydrate was detected in p27 and p68 by PAS staining. Papain treatment of p68 produced two distinct peptides, p27 and p22. Moreover, when an artificial mixture of p27 and p22 in a ratio of 1:1 was treated with 0.2 m-periodate for 30 min at 37 °C, only p22 was detectable. These results suggest that p68 is composed of p27 and p22, and that p27 is a glycosylated product of p22. Thus, from the evidence obtained, it is possible that p22 (22000 peptide) is the minimum size of the unique hepatitis B virus (HBV) gene product involved.
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The Sensitivity to γ-Irradiation of the Phases of the Virus-Host Interaction: Studies with Strains of Semliki Forest Virus in Mice
More LessSUMMARYA convenient and quantifiable model system, Semliki Forest virus (SFV) in the mouse, has been used to probe the wider problem of the radiation sensitivity of the distinct phases of the virus-host interaction. This has been investigated through the suppression and recovery of the several host defence mechanisms and cellular compartments involved. Direct observations have been made, following whole-body γ-irradiation at up to 600 R, of the sequential modifications imposed upon the efficiency of primary infection, the stimulation of regulatory immunity (pre-challenge) and the stimulation or boosting of protective immunity (post-challenge). These phases of the virus-host interaction show distinct sensitivities to γ-radiation which are discussed in terms of the impairment and recovery of the lymphocyte compartments probably involved.
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Confirmatory Evidence for the Association of Hepatitis B Surface Antigen with Antigenic Determinants Reactive with Antibodies Present in Some Anti-HBe-positive Sera
More LessSUMMARYSpherical 20 nm diam. particles of hepatitis B surface antigen (HBsAg), purified from some sera positive for hepatitis B e-antigen (HBeAg), are associated with antigenic determinants reacting with antibodies frequently present in sera containing anti-HBe. Treatment of HBsAg with the detergent Sarcosyl increased the exposure of these determinants which could be differentiated from HBeAg on the basis of immunological specificity and sensitivity to reduction and alkylation. The presence of these determinants appeared to be restricted to a subpopulation of HBsAg associated with IgG and albumin.
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Interferon Induction by Viruses. IV. Sindbis Virus: Early Passage Defective-Interfering Particles Induce Interferon
More LessSUMMARYWe have shown that a single defective-interfering (DI) particle of early (5th) passage Sindbis virus induces maximal amounts of interferon in an ‘aged’ primary chick embryo cell. The capacity of such DI particles to induce interferon is inactivated by small amounts of u.v. radiation (1/e dose = 232 ergs/mm2). The 1/e dose for inactivation of the interferon-inducing capacity of infectious virus particles is 399 ergs/mm2 and for infectivity is 1010 ergs/mm2. Pre-treatment with interferon blocks formation of interferon in response to either DI or infectious virus particles. Our results suggest that Sindbis virus genes must be expressed to form the interferon inducer, which is presumably a molecule of double-stranded (ds)RNA. We postulate that for interferon induction, the genomic RNA which codes for genes G and A must be translated into products whose concerted action produces a dsRNA molecule upon synthesis of a segment of RNA complementary to the genome. The RNA from early passage DI particles is sufficiently large (25S, 1.6 × 106 mol. wt.) to accommodate these genes, whereas the RNA from the late passage DI particles (20S, 1.0 × 106 mol. wt.) is not. Late (15th) passage DI particles do not induce interferon formation.
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Sindbis Virus RNA Replication. II. Strand Composition and Metabolic Fate of the Multi-stranded RNA Species
More LessSUMMARYDouble-stranded RNA from SB virus-infected cells was denatured and analysed on agarose-methylmercuric hydroxide gels. Equimolar amounts of three single-stranded species with mol. wt. of 4 × 106, 2.5 × 106 and 1.8 × 106 were found. Pulse and chase experiments in infected cells established a precursor-product relationship between the multi-stranded and single-stranded virus RNA species. The present results support the model in which the 49S and 26S species of virus RNA are synthesized in infected cells from two distinct replicating structures.
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Use of Hybridoma Monoclonal Antibodies in the Detection of Antigenic Differences Between Rabies and Rabies-related Virus Proteins. I. The Nucleocapsid Protein
More LessSUMMARYTwenty-one hybridoma cultures, obtained through the fusion of mouse myeloma cells with splenocytes of BALB/c mice immunized with either rabies virus or Mokola virus, secreted monoclonal antibodies specific for the nucleocapsid of the inducer virus. They displayed different specificities for the nucleocapsids of rabies and rabies-related viruses and could be classified into eight groups which are likely to correspond to different antigenic determinants on the nucleocapsid. Four strains of fixed rabies virus (CVS, ERA, Flury-LEP and Kelev) could not be differentiated by the nucleocapsid-specific hybridoma antibody. The Flury-HEP virus (derived from Flury-LEP) as well as the rabies-related viruses Mokola, Lagos bat and Duvenhage, showed marked differences in their reactivities with hybridoma antibodies to nucleocapsid. A selected panel of three of these hybridomas may be used for a rapid differential diagnosis among all members of the Lyssavirus group.
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Use of Hybridoma Monoclonal Antibodies in the Detection of Antigenic Differences Between Rabies and Rabies-related Virus Proteins. II. The Glycoprotein
More LessSUMMARYTwenty-five hybridoma cultures secreted monoclonal antibodies directed against the glycoprotein of rabies or rabies-related viruses. The antibodies had different specificities for the glycoproteins of eight rabies and rabies-related viruses. They could be classified into fourteen groups which probably correspond to different antigenic determinants on the glycoproteins. These hybridomas when used in either radioimmunoassay (RIA) or in neutralization tests allow differentiation of laboratory strains of rabies virus from each other as well as from the rabies-related viruses.
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Isolation of a Virus Closely Related to Gibbon Ape Leukaemia Virus from Cells Infected with Virus (HL-23V) Released by Human Leukaemic Cells
SUMMARYCanine thymus cells infected with virus (HL-23V) produced by human acute myelogenous leukaemia cells in culture were shown in previous reports to produce transforming and non-transforming type C virus similar or identical to the simian sarcoma virus complex SSV(SSAV) and to induce tumours in marmoset monkeys (Bergholz et al., 1977a). In these earlier studies the appearance of breakthrough foci at low dilutions of antiserum in neutralization tests with high-titred anti-SSV(SSAV) serum suggested the presence of another virus, distinct from SSV-(SSAV). We now report the isolation of this component and, by comparative neutralization analysis, demonstrate that it is most closely related to gibbon ape leukaemia virus (GALV). It is distinguished from SSV(SSAV) by kinetics of neutralization and molecular hybridization experiments. This component was readily cloned both from virus produced by HL-23V chronically-infected canine thymus cells established by Teich et al., (1975) when HL-23V was first isolated and from virus produced by HL-23V-induced marmoset tumour cells in culture. The presence of this component in the original leukaemic cell cultures is discussed.
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Integration and Transcription of Virus DNA in Herpes Simplex Virus Transformed Cell Lines
More LessSUMMARYThe physical state of the HSV 1 DNA present in two biochemically transformed cell lines, a revertant line and a supertransformed cell line, was determined. These cells all contained fragments of the HSV genome and the transformed and super-transformed cell lines expressed virus thymidine kinase. It was found that the virus DNA in these cells was maintained in a complex state with approximately half of the HSV DNA present in a covalently integrated state and the other half in a non-integrated state. There was no major cell line difference in the distribution of integrated and non-integrated virus DNA. RNA transcripts representing 5% of the HSV 1 genome are present in each of these lines. This is more than is required to code for the virus thymidine kinase present in the transformed and supertransformed cell lines and suggests the presence of other virus proteins in these cells.
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Deletions of the Terminal Sequences in the Genomes of the White Pock (u) and Host-restricted (p) Mutants of Rabbitpox Virus
More LessSUMMARYThe DNAs of wild-type rabbitpox virus (u +.p +), and selected u (white pock, u.p +) and p (white pock, PK-15 cell non-permissive, u.p.) mutants were compared by restriction enzyme analysis. The cleavage fragments produced by digestion with HindIII or EcoRI endonucleases were separated by agarose gel electrophoresis. Deletions of approx. 10 × 106 were recognized at the right-hand terminus of each u mutant (three isolates), whereas deletions of 5 × 106 to 20 × 106 were detected at the left-hand terminus of each p mutant (nine isolates). Rapidly renaturing terminal restriction fragments, indicative of the covalent cross-links, were only found at the terminus unaffected by deleted sequences. Recombinant viruses that were wild-type in both genetic characters and in restriction pattern were recovered from mixed infections involving u and p mutants, but no genetic interaction was detected between crosses involving only u or p mutants. The p mutants were divided into two non-permissive classes dependent on their biochemical expression in PK-15 cells: the ‘early’ class which failed to replicate virus DNA and synthesized only the ‘early’ polypeptides and the ‘late’ class which appeared to be normal both in virus DNA and polypeptide synthesis. There was no correlation between the extent of the left-hand terminal deletion and the extent of permissiveness of the p mutants in PK-15 cells.
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A Comparison of Measles and Canine Distemper Virus Polypeptides
More LessSUMMARYThe polypeptides induced by canine distemper virus (CDV) strains have been characterized by polyacrylamide slab gel electrophoresis of infected cell lysates labelled with 35S-methionine, 14C-amino acids, 3H-glucosamine and 3H-mannose, or 32P-orthophosphate. Seven virus-induced polypeptides have been assigned the following nomenclature and mol. wt.: a large polypeptide L (180000); a large glycoprotein H (77000); a nucleocapsid-associated protein P (73000); the nucleocapsid protein N (60000); the smaller glycoprotein F0 (59000); a membrane protein M (35000) and a small polypeptide S (15000). During pulse-chase experiments with 3H-glucosamine and 14C-amino acids the intensity of the F0 band decreases and that of the F1 and F2 bands increases; the H polypeptide band becomes more diffuse and the S-protein disappears. The N- and P- but not the M-proteins have been found to be phosphorylated. The polypeptide pattern of the Onderstepoort strain of CDV has been compared with that of two other CDV and with 17 measles and subacute sclerosing panencephalitis (SSPE) strains. Differences in the mobilities of various polypeptides have been observed between CDV and measles and SSPE strains; however, the only consistent difference is the mol. wt. of the M-protein of CDV strains which is smaller by 2000 than that of MV and this may be a biochemical marker to distinguish CDV from measles and SSPE virus strains.
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DNA Sequence Homology Relationships Among Six Lepidopteran Nuclear Polyhedrosis Viruses
More LessSUMMARYThe DNA sequence homology relationships among six lepidopteran nuclear polyhedrosis viruses (NPVs) have been explored by hybridization of 32P-labelled NPV DNAs to Southern blots of restriction endonuclease-digested NPV DNA. The Autographa californica NPV (AcMNPV) shows extensive DNA sequence homology throughout the entire genome with the Rachiplusia ou NPV (RoMNPV). Both the Orgyia pseudotsugata NPV (OpMNPV) and the Porthetria dispar NPV (PdMNPV) share homologous regions, equivalent to 1% of the DNA genome, with AcMNPV and RoMNPV. This homology is localized in two regions on the AcMNPV physical map although other regions are also weakly homologous. Approx. 1% of the DNA of OpMNPV and PdMNPV show sequence homology with each other; the homology is primarily localized in two to four regions of the genomes. Heliothis zea NPV (HzSNPV) and Trichoplusia ni NPV (TnSNPV) share less than 0.2% sequence homology with the MNPVs and share less than 0.2% sequence homology with each other.
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Infectivity and Pathogenicity of Nodamura Virus for Mosquitoes
More LessSUMMARYNodamura virus multiplied and caused paralysis and death when injected into the thoraces of adult Aedes albopictus and Toxorhynchites amboinensis mosquitoes but not when similarly injected into Culex quinquefasciatus adults. A. albopictus also became infected after ingesting a Nodamura virus suspension or after immersion in a virus suspension as larvae, but they did not die. Head squash preparations of the injected insects, examined by indirect fluorescent antibody technique, showed large amounts of Nodamura virus antigen in the brain regardless of the mode of infection. Nodamura virus was isolated from and titrated in mosquito cell (AP-61) cultures. Comparative titrations indicate that this method of assay is more sensitive than intracerebral inoculation of infant mice.
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Polyamine Metabolism in MRC-5 Cells Infected with Human Cytomegalovirus
More LessSUMMARYThe rate of putrescine uptake into MRC-5 cells increased markedly following infection with human cytomegalovirus (HCMV). Enhanced incorporation occurred immediately after infection and the highest levels were attained following the production of infectious, progeny virus. Parallel kinetic changes in the utilization of radio-labelled putrescine were shown by the amounts of spermidine and spermine recovered from infected cells as radioactive derivatives. A temporal correlation was found between these changes in polyamine metabolism and the synthesis of virus DNA. Methylglyoxalbis(guanylhydrazone), an inhibitor of spermidine and spermine synthesis, did not affect virus replication if HCMV-infected cells were exposed to the inhibitor after completion of the eclipse phase of the virus growth cycle. These results show that polyamine metabolism is required only during the initial stages of HCMV replication.
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Characterization and Isolation of Structural Polypeptides in Haemagglutinating Encephalomyelitis Virus
More LessSUMMARYHaemagglutinating encephalomyelitis virus (HEV), a member of the coronavirus family, was purified and analysed by SDS-polyacrylamide gel electrophoresis. It was shown to contain eight polypeptides, seven of which were glycosylated. They had apparent mol. wt. of 180000 (GP 180), 130000 (GP 130), 120000 (GP 120) 76000 (GP 76), 64000 (VP 64), 54000 (GP 54), 32000 (GP 32) and 31000 (GP 31). Electrophoresis of virus samples dissociated under varying conditions showed that GP 54 and GP 120 could be interpreted as larger products of GP 31 and GP 32 and of GP 76, respectively. GP 76 also appeared as a dimer with a mol. wt. of 140000 (GP 140) in the absence of β-mercaptoethanol. Subviral particles, obtained by treatment with bromelain, banded at a slightly lower density than the intact virus and lacked surface projections. Analysis of these particles indicated that GP 180, GP 130 and GP 76 are associated with the virus projections. A small part of GP 31 and GP 32 also appeared to protrude from the lipid envelope, since 20% of each molecule was sensitive to digestion. Two glycoproteins, GP 130 and GP 76, were solubilized with the detergent Triton X-100 and separated by rate zonal centrifugation. According to its activity in indirect haemagglutination tests, GP 76 was considered to be a monovalent haemagglutinin subunit.
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Phosphorylation of Iridescent Virus Polypeptides in vitro
More LessSUMMARYIridescent virus types 22 and 23 possess a protein kinase which phosphorylates most, but not all, of the virus structural polypeptides in vitro.
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The Polypeptide Composition of Isolated Surface Projections of Avian Infectious Bronchitis Virus
More LessSUMMARYDisruption of avian infectious bronchitis virus (IBV) particles with 4% Triton X-100 and 1.0 m-KCl and centrifugation through a sucrose gradient containing 0.1% Triton X-100 and 1.0 m-KCl enabled separation of the petal-shaped surface projections. By negative-contrast electron microscopy the separated projections appeared mainly as rosettes containing 3 to 12 projections radiating from a central core, although single projections and rosettes containing up to 16 projections were seen. SDS-PAGE of these preparations revealed two polypeptides of 86000 and 66000 mol. wt. The larger polypeptide was glycosylated.
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Recombination in Adenovirus. I. Analysis of Recombinant Viruses Under Non-selective Conditions
More LessSUMMARYA method is described, based on an approach previously employed for the mapping of adenovirus ts mutants (Grodzicker et al., 1975), for the identification of recombinant viruses in the progeny of mixed infections of human HeLa cells with wild-type adenovirus types 2 and 5 under non-selective conditions. Differences in restriction enzyme sites for the endonuclease HpaI could be used to detect new recombinant DNA fragments in a yield of 3 to 6% after single mixed infections and yields up to 25% following three successive mixed infections. The recombinant nature of the new fragments was proved by the clonal isolation of recombinant viruses in similar yields. Analysis of one cloned recombinant pointed to the occurrence of multiple crossover events at different sites along the virus chromosome. The results are discussed in terms of a biochemical approach towards the study of the mechanism of general genetic recombination in human cells as well as with respect to the evolution of adenoviruses.
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