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Volume 35,
Issue 3,
1977
Volume 35, Issue 3, 1977
- Articles
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Morphology and Stability of Infantile Gastroenteritis Virus: Comparison with Reovirus and Bluetongue Virus
More LessSUMMARYThree forms of infantile gastroenteritis virus (IGV), each representing the exposure of a different surface layer, were studied by negative-contrast electron microscopy and compared with analogous forms of a reovirus and an orbivirus. Although major structural similarities were found in each of the three main layers of these viruses, differences were sufficient to clearly distinguish the member genera of the Reoviridae family. IGV particle morphology was stable to most physical and chemical agents commonly used to evaluate virus lability. Virions were stable under all test conditions except when treated with versene-trypsin or held at pH < 3.
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Characterization of the RNA Mycovirus Infecting Allomyces arbuscula
More LessSUMMARYThe mycovirus infecting Allomyces arbuscula has been fractionated into two particle classes designated A and B. Particles A have a buoyant density of 1.385 g/cm3, in CsCl, a sedimentation coefficient of 75S and contain one major and two minor polypeptides with molecular weights of 34000, 31000 and 28000, respectively. Particles B band at 1.335 g/cm3, sediment with a value of 67S and, in addition to the three polypeptides as in particles A, they contain seven extra polypeptides, six of which are of lower molecular weights. Both particles A and B contain double-stranded RNA which was resolved by electrophoresis on polyacrylamide gels into three components with molecular weights of 1.4 (major), 2.5 and 1.1 × 106 (minor). Both particles also contain single-stranded RNA. Particles A contain 27% RNA whereas particles B only contain 14%. Evidence is presented that particles A and B belong to the same type of virus and represent two different replicative stages.
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Isoelectric Focusing Studies of Mengo Virus Variants, their Protein Structure Units and Constituent Polypeptides
More LessSUMMARYVirions of three plaque variants (L-, M- and S-) of Mengo virus, which assume a bimodal distribution when subjected to isoelectric focusing in sucrose-stabilized pH gradients containing 0.5% Brij 35, were found to band in a single peak (at pH 8.1 to 8.4) when focused under the same conditions in gradients supplemented with 6 m-ethylene glycol. An examination of m-Mengo virions, isoelectric at pHs 4.7 and 8.4 in sucrose-stabilized gradients, showed that the two populations were indistinguishable on the basis of specific infectivity, polypeptide composition and sedimentation characteristics, but differed in their ability to agglutinate human erythrocytes.
When pH-inactivated virions were subjected to isoelectric focusing, three well defined peaks were produced, one of which was found to correspond to the well characterized 13.4S subunit of Mengo virus, and the other two — on the basis of compositional analysis — to virions that had lost some of their 13.4S structure units. No difference in electrophoretic behaviour was found among the 13.4S subunits isolated from the three Mengo variants — all three were found to be isoelectric at pH 5.85 to 6.00. Analysis, by isoelectric focusing, of the four structural polypeptides (α, β, γ, δ) isolated from L-, M- and S-Mengo failed to reveal any significant differences among the three variants.
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The 50S and 35S RNAs from Newcastle Disease Virus-Infected Cells
More LessSUMMARYElectrophoretic analyses of the 50S and 35S Newcastle disease virus-specific RNAs from infected cells before and after heat denaturation make it possible to demonstrate that these regions each contain single-stranded RNAs with corresponding S values as well as partially base-paired structures. The partially base-paired structures which sediment at 50S (40 to 60S) have a distribution in gels similar to that of the in vitro transcriptive intermediates, and they remain when 50S RNA synthesis (replication) is blocked by cycloheximide. The partially base-paired 35S RNA is more homogeneous and is neither labelled in the in vitro transcription reaction nor when infected cells are treated with cycloheximide. These base-paired structures may, therefore, be involved in transcription and replication, respectively.
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A Comparative Study of Related Alphaviruses — a Naturally Occurring Model of Antigenic Variation in the Getah Sub-group
More LessSUMMARYThe relative susceptibilities of six cell culture lines were compared using six closely related alphaviruses. Plaque morphology was used as a parameter for biological differences of these viruses in parallel with plaque reduction neutralization tests. One virus showing mixed plaque sizes was plaque purified and the two variants thus obtained were compared by in vivo and in vitro methods. The implications of plaque variants within a mixed virus population are considered along with possible natural selective mechanisms in the evolution of these related viruses of synpatric distribution.
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Single Radial Immunodiffusion Test for Detecting Antibodies Against Surface Antigens of Intracellular and Extracellular Vaccinia Virus
More LessSUMMARYAntibodies to surface antigens of intracellular naked vaccinia virus (INV) and in limited studies extracellular enveloped virus (EEV) were determined by single radial immunodiffusion tests (SRDT) with immobilized virions in agarose gels. Antibodies to INV were demonstrable in rabbit hyperimmune sera (one to four visible zones), smallpox convalescent sera and sera from re-vaccinated individuals. A difference in specificity of antibodies reacting with INV and EEV was detectable by SRDT. The SRDT provides a simple, reproducible and specific test for detection of antibodies against vaccinia or variola virus, but the technique requires large quantities of virions.
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Oncogenic Transformation of Rat Embryo Fibroblasts with Photoinactivated Herpes Simplex Virus: Rapid In Vitro Cloning of Transformed Cells
More LessSUMMARYRat embryo fibroblasts (REF) were transformed in vitro with photoinactivated herpes simplex virus. Low passage (7 to 10) HSV-transformed rat cells (t-REF-line G) produced multiple tumours in 49% of newborn rats with a latent period of 20 to 24 weeks. An in vitro cloning procedure for transformants in the uncloned t-REF-line G cells produced clonal lines which varied from non-oncogenic to clonal lines producing tumours with shorter latent periods (10 to 14 weeks) compared to uncloned cells. At passage 30, t-REF-line G-clone 1 cells produced rapidly growing tumours in 100% of the newborn rats with a latent period of only 2 to 3 weeks. Tumour cells (RFS 12-22-75) established in culture produced tumours within 2 weeks after subcutaneous (s.c.) inoculation of weanling rats (100% with tumours) and they were transplantable to 100% of inoculated adult rats. Histopathological examination of all tumours produced in newborn, weanling or adult rats revealed large, poorly differentiated malignant fibrosarcomas; metastatic tumours were observed in the lungs of 10 to 20% of newborn rats inoculated s.c. with RSF cells. Approx. 25 to 50% of the clonal transformed or tumour cells synthesized HSV-specific-antigens detected by immunofluorescence. HSV-transformed and tumour cells are resistent to superinfection by the homologous transforming virus. Since the in vitro cloning procedure for transformant cells can readily segregate cells producing clonal lines varying in oncogenic potential, the procedure might have useful application in elucidating HSV oncogenesis.
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Cell Generation and Type C Virus Expression in the Human Embryonic Cell Strain HEL-12
More LessSUMMARYThe spontaneous expression of a type C virus in a diploid strain of human embryonic lung fibroblast-like cells (HEL-12) was examined during serial culture. Virus antigen expression was determined by indirect immunofluorescence with antisera to disrupted simian sarcoma virus and the 28000 mol. wt. internal antigen of the endogenous cat virus RD-114. Virus production was examined by reverse transcriptase assays of culture fluids. Virus antigens were not detected for 25 days after frozen, primary HEL-12 cells were reinstated in culture. The cells expressed virus antigens but did not release virus particles between 25 and 80 days. Spontaneous virus release and maximal antigen expression occurred in cells grown for 80 to 120 days. Virus particles were not detected after 120 days although virus antigens persisted until the experiment was terminated. The HEL-12 virus was infectious for cell cultures of human, rhesus monkey, dog and rabbit cells. The proportion of SiSV-like and RD-114-like antigenic components of HEL-12 virus were altered by passage through heterologous cells suggesting heterogeneity of the HEL-12 virus population.
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Polyadenylic Acid [Poly(A)] Sequences Associated with Measles Virus Intracellular Ribonucleic Acid (RNA) Species
More LessSUMMARYThe kinetics of 3H-uridine incorporation into measles-infected Vero cells demonstrated that maximum virus-specific RNA synthesis occurred between 16 and 20 h after infection. Sedimentation analysis on sucrose gradients revealed the presence of four species of RNA having sedimentation coefficients of 4S, 12 to 26S, 28 to 36S, and 50S. Annealing studies showed that RNA sedimenting in the 12 to 36S regions was 100% complementary in base sequence to nucleocapsid 50S RNA, and at least 96% of the 50S genomic RNA was transcribed during virus replication. Polynucleotide binding experiments and ribonuclease treatment indicated that poly(A) sequences were associated with the intracellular 12 to 26S, 28 to 36S and 50S RNAs. Denaturation of intracellular 50S RNA followed by sucrose gradient centrifugation demonstrated that this was a mixture of genomic 50S and heterogeneous RNAs which sedimented at 4 to 40S. The genomic RNA did not contain poly(A) sequences, and these are presumably associated with the heterogeneously sedimenting RNAs. The size of poly(A) sequences present on the 12 to 36S RNAs was estimated to be in the range of 70 to 140 nucleotides. Treatment of the 12 to 36S RNAs and their poly(A) sequences with polynucleotide phosphorylase indicated that the poly(A) was located on the 3′ end of the RNAs, but that under the experimental conditions used this was protected by the secondary structure of the molecules.
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Expression of Feline Leukaemia Virus Antigens on Cat Lymphoma Cells: Kinetics of Biosynthesis
More LessSUMMARYCultured feline lymphoma cells (FL-74), productively infected with a feline leukaemia virus (FeLV) were quantitatively examined with a radioimmune assay for cell membrane associated FeLV-p27 and total FeLV associated cell surface antigens (FeLV-CSA) using a monospecific antiserum and a broadly reactive antiserum respectively. The infected cells bound 5.6 × 105 anti-FeLV-p27 IgG molecules/cell, representing 40% of the total FeLV-CSA detected. The kinetics of synthesis of surface associated p27 and total FeLV-CSA was determined following their removal by treatment of intact cells with trypsin. Both p27 and the total FeLV-CSA population reappeared on the cell surface within 6 to 8 h following trypsin digestion. Antigen re-expression was blocked by cycloheximide but not by actinomycin D or cordycepin. The time course of antigen decay with these same antimetabolites indicated that the average turnover rate of cell surface p27 and FeLV-CSA was 6 to 8 h while the mRNAs which specify these antigens have a lifetime of at least 10 h. Virus production was blocked in less than 2 h by cycloheximide, and within 2 to 4 h by actinomycin D. Virus production continued at a reduced rate for at least 6 h in the presence of cordycepin. The difference in sensitivity to inhibitors of RNA synthesis of p27 and FeLV-CSA production (blocked in 9 to 10 h) and of virus production (blocked in 2 to 4 h) supports the proposition of two non-equilibrating pools of intracellular virus RNA molecules with different half-lives: one associated with polyribosomes, and another which becomes encapsulated in the completed virion.
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Metabolism of Interferon: Hepatic Clearance of Native and Desialylated Interferon
More LessSUMMARYThe effect of removal of sialic acid on the survival of rabbit serum and urinary interferon (IF) has been investigated in isolated, perfused rabbit liver preparations. In contrast with native IF, which may be already partially desialylated, IF freed of 80 to 90% of its sialic acid was rapidly cleared from the perfusate of normal livers, or livers pre-treated with actinomycin D. The results suggest that the mechanism of IF catabolism by the liver is similar to that reported for several other circulating glycoproteins.
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An Ultrastructural Study of Inclusions and Disease Development in Plant Cells Infected by Cowpea Chlorotic Mottle Virus
More LessSUMMARYSubcellular responses of leaf cells to infection by bean yellow stipple virus, a strain of cowpea chlorotic mottle virus (CCMV), in two varieties of bean, Phaseolus vulgaris L., and three varieties of cowpea, Vigna sinensis (Torner) Savi, were investigated by electron microscopy. Amorphous inclusions (AI), filamentous inclusions (FI), which appeared to be characteristic of CCMV infection, and membranous vesicles (30 to 100 nm in diam.), containing fibrils and virus particles, were consistently found in cells in all infected hosts. The AI occurred in cytoplasm as granular masses sandwiched between the rough endoplasmic reticulum (RER) and the nuclear envelope (NE) at an early stage of infection. In later samples AI were scattered randomly in masses of various sizes, numbers and shapes. Electron lucent areas containing virus particles were present in large masses of AI. The FI were long, unbranched, flexuous, rod-like structures of 17 nm in diam. They appeared after AI were formed in the cytoplasm and also occurred in the nucleus. Cytoplasmic FI were usually associated with AI, and nuclear FI with the nucleolus. Intranuclear virus particles were observed only when FI were located in the nuclei. Both FI and AI were similarly degraded by treatment with subtilisin or pronase, but virus particles were not. Fibril-containing vesicles arising from the membranes of NE and RER were concurrently present in the perinuclear space and the lumen of ER associated with AI and FI.
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Complementation of Adeno-Associated Satellite Virus (AAV) by Temperature-Sensitive Mutants of Adenovirus Type 31
More LessSUMMARYTemperature-sensitive (ts) mutants of human adenovirus type 31 were able to complement adeno-associated satellite virus (AAV) antigen production in both HEK and KB cells at both permissive and non-permissive temperatures. However, mutant ts 94, an adenovirus 31 mutant which produces apparently normal amounts of structural protein and DNA but is defective in maturation, was significantly inhibited in its ability to potentiate AAV infectivity at the non-permissive temperature. Normal AAV DNA and adenovirus DNA were isolated from co-infections with AAV and mutant ts 94 at the non-permissive temperature. We suggest that an adenovirus-coded maturation function common to both adenovirus and AAV maturation is defective in the ts 94 system.
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An Extended Genetic Recombination Map for Foot-and-Mouth Disease Virus
More LessSUMMARYThe original foot-and-mouth disease virus recombination map (Lake, Priston & Slade, 1975), which included 35 mutagen-induced ts mutants, has been extended both in detail and size by the mapping of a further 33 ts mutants (9 mutagen-induced and 24 spontaneous). The size increase from 0.57% to 3.27% maximum recombination frequency was principally due to the use of a new standardization technique for recombination frequencies but, in addition, the original map distance was increased by approx. 30% due to the mapping of new mutations. As in the original map, there was a marked concentration of mutations near the guanidine (gs) locus, i.e. 83% of the mutants had mutations in the third of the map adjacent to the gs locus.
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Isatin-β-Thiosemicarbazone Causes Premature Cessation of Vaccinia Virus-Induced Late Post-Replicative Polypeptide Synthesis
More LessSUMMARYThe effects of isatin-β-thiosemicarbazone on vaccinia virus-induced polypeptide synthesis has been examined by polyacrylamide gel electrophoresis and autoradiography. The synthesis of pre-replicative and early post-replicative polypeptides proceeded normally in the presence of the drug; the onset of late post-replicative polypeptide synthesis occurred with normal timing under these conditions but the rate of synthesis of all virus polypeptides then declined rapidly.
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Is Interferon Tissue Specific? — Effect of Human Leukocyte and Fibroblast Interferons on the Growth of Lymphoblastoid and Osteosarcoma Cell Lines
More LessSUMMARYThe growth inhibitory effect of human leukocyte and fibroblast interferons was tested in vitro. The effect of fibroblast interferon was more pronounced on osteosarcoma cells and the effect of leukocyte interferon was more pronounced on lymphoid cells. This suggests that the capacity of interferon to inhibit cell growth is, in some measure, tissue specific.
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The Effect of Cycloheximide on the Replication of Measles Virus
More LessSUMMARYTreatment of measles virus-infected cells with cycloheximide results in a three-fold increase of 3H-uridine incorporation into the 12 to 36S mRNA species and in the inhibition of genomic 50S RNA synthesis. Consistent with these observations was the finding of a build-up of polyribosomes but an absence of nucleocapsids in the infected cells. These results suggest that measles virus RNA replication, but not transcription, is dependent upon active protein synthesis.
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Demonstration of Size Variation of RNA Segments Between Different Isolates of Calf Rotavirus
More LessSUMMARYPolyacrylamide gel electrophoresis of RNA extracted from wild rotavirus isolates and cell culture-adapted virus revealed a significant variation in the molecular weight of individual RNA segments. The major differences were observed between wild isolates on the one hand and the adapted strain on the other hand. The slight variations that were observed between different wild isolates were found regularly and appeared to be related to the origin of the samples.
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Replication of Minute Virus of Mice in Chinese Hamster Ovary Fibroblasts
More LessSUMMARYMinute virus of mice (MVM), a parvovirus, replicates in Chinese hamster ovary (CHO) cells. Although replication of the virus cannot readily be detected by haemagglutination, it can be measured by plaque assay on mouse strain LM cells.
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A Sensitive and Easy Local Lesion Assay for Turnip Yellow Mosaic Virus
More LessSUMMARYChinese cabbage plants grown in soil without the addition of nutrients developed local lesions after inoculation with turnip yellow mosaic virus or its RNA, provided that the inoculated leaves were illuminated by mercury-vapour lamps. A linear relationship was found between lesion number and concentration of RNA.
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