- Volume 21, Issue 1, 1973
Volume 21, Issue 1, 1973
- Articles
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Interferon Bioassay: Reduction in Yield of Myxovirus Neuraminidases
More LessSUMMARYInterferon reduced the production of virus neuraminidase during single-cycle growth of A2/HK/1/68 or recombinant X7(F1) influenza viruses in chicken embryo cell cultures. Neuraminidase activity, measurable within 6 h after infection, was reduced as much as 70 to 80% below control levels in interferon-treated cultures. X7(F1) neuraminidase yield was at least as sensitive a measure of interferon inhibition as vesicular stomatitis virus in the standard plaque-reduction assay. Interferon titre, expressed as the 30% neuraminidase reduction dose or NRD30, is derived from semi-logarithmic sigmoidal dose—response curves. The advantages that recommend the neuraminidase reduction bioassay for interferon assay include the precision of the enzyme assay (±1.0%), the precision of measurement of replicate samples of interferon (±9%), the reproducibility of interferon titres obtained sequentially (±20%), as well as the rapidity and economy of the method.
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Mitochondrial Vesiculation Associated with Cucumber Green Mottle Mosaic Virus-infected Plants
T. Hatta and R. UshiyamaSUMMARYCells of various tissues, such as leaves, roots, flowers, and fruits, from Cucumis sativus L. plants infected with cucumber green mottle mosaic virus (CGMMV) have been studied by electron microscopy. Virus particles were consistently observed in the ground cytoplasm of various tissue cells infected with CGMMV. The small vesicles observed in the mitochondria were probably a characteristic modification related to the virus replication. They were always bounded by a single membrane and located in the perimitochondrial spaces between outer and inner membrane, and in the cristae. These vesicles could be formed in the mitochondria as soon as or before virus production begins, and they seemed to be increased with the development of virus infection.
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Virus-specified Polypeptides in Cells Infected with Semliki Forest Virus
More LessSUMMARYFive polypeptide components were resolved by polyacrylamide gel electrophoresis of the proteins extracted from cells infected with Semliki Forest virus. One of these components was the virus nucleocapsid polypeptide, another was the two unresolved virus envelope glycoproteins. The remaining three peaks were due to polypeptides of mol. wt. 97000 (NVP-97), 78000 (NVP-78) and 63000 (NVP-63). NVP-97 and NVP-63 are glycoproteins, and both NVP-63 and the envelope glycoproteins were precipitated with either an antiserum to the envelope polypeptides or with concanavalin A.
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Interferon Induction Site: Poly IC on Solid Substrate Carriers
More LessSUMMARYThe complex of polyinosinate with polycytidylate (poly IC) has been attached to solid carriers which can be suspended in the medium over the cell monolayer; it has also been attached to surfaces which may be overgrown by the cell monolayer. Interferon production and antivirus protection achieved by these inducer systems were measured and it was found that such anchored poly IC conveyed antivirus protection. However, in all systems studied, some release of polynucleotide from the carrier was observed and thus the results do not prove that mere contact of inducer with the external membrane is sufficient for induction of interferon.
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Size Differences in the Ribonucleic Acids of Feline Leukaemia Viruses
More LessSUMMARYElectrophoretic analysis of native and denatured RNA of feline leukaemia virus (FeLV) has revealed small but significant size differences between the RNAs of a number of FeLV isolates. The mol. wt. of the denatured or subunit RNA has been estimated to range from 2.2 to 2.6 × 106 in the different isolates, each of which belonged to one or more FeLV subgroups (A, B and C). The RNA of subgroup A virus was smaller than that of subgroup B virus, while the RNA of subgroup C virus appeared to be intermediate in size between the RNAs of A and B subgroup viruses. Size differences were, however, also found between RNAs of FeLV isolates of a single subgroup (A), indicating that no simple relationship exists between RNA size and virus subgroup.
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Transfection of Chicken Fibroblasts with Single Exposure to DNA from Virogenic Mammalian Cells
More LessSUMMARYThe efficiency of the transfection of chicken fibroblasts with a single dose (1.5 to 150 µg) of DNA isolated from virogenic RSV (Prague strain) transformed XC cells was increased if chicken fibroblasts were pre-treated with BUdR. Mitomycin or u.v. irradiation in doses used were not efficient. The repeated attempts to transfect duck fibroblasts, not containing activable endogenous chicken virus genome or group-specific antigen, always failed.
Both DNA isolated from purified nuclei and whole virogenic cells exerted the same transfecting activity.
Transfecting activity was present in the peak fractions obtained after CsCl gradient sedimentation of DNA obtained from virogenic cells, was absent in RNA preparations and after digestion of DNA with DNase or alkaline denaturation. This indicates that DNA is responsible for transfection. The role of endogenous virus genomes in transfection is discussed.
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Quantitative Studies of Oncornaviruses in Thin Sections
More LessSUMMARYA thin sectioning procedure was used to detect and enumerate oncornavirus particles in tumour-cell culture fluids, tumour homogenates, mouse blood plasma, mouse and human milk specimens, and density gradient fractions of these specimens. Oncornaviruses studied included mouse mammary tumour, murine sarcoma, ESP-1, and RD-114 viruses. In this procedure particles were sedimented on to small membrane filter discs in the ultracentrifuge using inexpensive commercially available adapters and tubes. Particles in cross sections of the disc were counted and their total number determined by relating the effective surface area of a field to the surface area of the entire membrane disc. Particles may be reliably identified and counted in preparations containing predominately cellular debris. Linear dose response plots were obtained in serial dilution experiments utilising vaccinia virus, adenovirus type 2, and murine sarcoma virus, demonstrating the reliability of the procedure and its wide applicability. The counts obtained for adenovirus and vaccinia virus preparations were comparable to counts obtained for the same preparations in other laboratories by established methods. Statistically reliable counts have been obtained using sample vol. of 0.01 ml or less.
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Studies on a Virogenic Clone of SV 40-transformed Rabbit Cells Using Cell Fusion and in situ Hybridization
More LessSUMMARYA clone of baby rabbit kidney cells transformed with SV 40 virus was found to be releasing virus at the rate of about 1 infectious unit per 104 cells per day. Virus antigen was detected in between 1 in 103 and 1 in 104 cells. In situ hybridization with radioactive SV 40 complementary RNA revealed that about 3% of the transformed cells were producing virus DNA. When the transformed cells were fused with permissive cells and incubated in anti-SV 40 serum between 2% and 8% of the heterokaryons formed produced SV 40 virus. A model is proposed according to which the cells in this clone, and possibly other transformed clones, may exist in one of three states: (A) the commonest state, in which virus DNA is not replicable autonomously; (B) a state in which virus DNA can be replicated autonomously, but late virus proteins are not made; and (C) a state in which complete virus can be synthesized.
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Sequential Reconstitution of Tobacco Rattle Virus
More LessSUMMARYConditions suitable for in vitro reconstitution of the CAM strain of TRV have been defined, and various steps of rod morphogenesis have been studied.
The CAM strain of TRV was found to reconstitute best at pH 4.7 in 0.5 m-phosphate buffer at low temperature, unlike the C isolate of TRV which was shown by Semancik & Reynolds (1969) to reconstitute partially at pH 8.0 in glycine buffer.
While the initiation step consisting of the binding of a RNA molecule to a 36 S double layer protein disk took place under wide pH and ionic strength ranges, elongation proved to be a much more refined process which required well-defined conditions of pH, ionic strength and temperature.
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The Formation of Poliovirus Particles in Association with the RNA Replication Complexes
More LessSUMMARYPoliovirus particles are associated with the RNA replication complexes and can be identified by velocity sedimentation and electron microscopy. After pulse-labelling with [3H]-uridine beginning 3.5 h after infection, these virus particles have a radioactivity to infectivity ratio three- to eightfold higher than virus particles from other cell fractions. A specific association between the RNA replication complexes, poliovirus particles and smooth cytoplasmic membranes is shown by isopycnic sedimentation and partial resistance to enzyme digestion. These results strongly suggest that virus RNA replication and particle formation are coupled processes which occur in association with smooth cytoplasmic membranes.
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Ross River Virus Replication in Cultured Mosquito and Mammalian Cells: Virus Growth and Correlated Ultrastructural Changes
More LessSUMMARYThe growth of Ross River virus in cultured mosquito (Aedes albopictus) and monkey kidney (Vero) cells shows similar latent periods (5 to 6 h) and maximum yields.
In Aedes albopictus cells the virus establishes a persistent, non-cytopathic infection with no significant change in cell division rate. Virus matures within large, electron-dense cytoplasmic inclusions and also at the cell membrane. Virus accumulates within the inclusions but nucleocapsids do not. ‘Type-1 cytopathic vacuoles’ (Grimley, Berezesky & Friedman, 1968) are not found. Between 40 and 60 h after infection, the cell-associated virus titre falls by 1 log unit. Cytoplasmic inclusions lose electron-dense material and are transformed into microvesiculated vacuoles. Virus progressively disappears from these structures and from the cell membrane. It is suggested that during the establishment of the persistent infection, digestion of the contents of the inclusions occurs, resulting from fusion of lysosomal microvesicles with the inclusions.
In Vero cells infection leads to cell lysis. Early in infection virus is found in small cytoplasmic vesicles: ‘type-1 cytopathic vacuoles’ are also present. Accumulation of nucleocapsids is marked, particularly late in infection. Thus, although the pathogenesis of Ross River virus in mice is atypical (Mims et al., 1973; Murphy et al., 1973), its ultrastructural development is similar to that of other alphaviruses in cultured vertebrate cells.
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The Interaction of Herpes Simplex Virus with Cultures of Peripheral Nervous Tissue: an Electron Microscopic Study
More LessSUMMARYStrains of herpes simplex virus types 1 and 2 were used to infect 3-day-old cultures of chick embryo dorsal root ganglia. Evidence of c.p.e. was seen by means of light microscopy at 14 h after infection. Ganglia were removed at intervals after infection and fixed and sectioned for electron microscopy. All stages of virus replication were observed in neurons, while few glial cells showed evidence of infection. Glial cells which did appear to have become infected showed signs of defective virus replication. The significance of the findings are discussed in relation to the mechanism of latency in herpes simplex virus infections.
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Synthesis of Virus-capsid Antigen (VCA) Enhanced by Ultraviolet Irradiation of a Lymphoblastoid Cell Line carrying Epstein–Barr Virus
More LessSUMMARYU.v. irradiation of SH-RP cells, a lymphoblastoid cell line carrying Epstein–Barr virus, which was derived from a patient with acute myeloid leukaemia, retarded cell growth but significantly enhanced the percentage of cells containing virus-capsid antigen (VCA) as demonstrated by indirect immunofluorescence. The effects of u.v. irradiation on SH-RP cultures were reversible under normal conditions of medium replenishment every four days. Starving the cultures did not modify these effects.
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Virus-like Particles Associated with Two Diseases of Colocasia esculenta (L.) Schott in the Solomon Islands
More LessSUMMARYBacilliform virus-like particles of two sizes are found associated with the diseases ‘Alomae’ and ‘Bobone’ of Colocasia esculenta in the Solomon Islands. Neither kind of particle was transmitted mechanically or by aphids. The smaller particles were similar in size and shape to cocoa swollen-shoot virus and measured 125 × 28 to 29 nm in negative stain. In partially purified preparations their sedimentation coefficient was 285 S. In thin sections of diseased C. esculenta they were found in the cytoplasm of phloem sieve tubes loosely aggregated with amorphous material, or packed closely, but haphazardly, in bundles. The larger particles were found in sieve tubes, companion cells and mesophyll. They measured 300 to 335 × 50 to 55 nm and in transverse sections showed three electron-dense layers, a central core about 9 nm in diam., an inner annulus approx. 28 nm thick and an outer annulus 3 to 5 nm thick. Large particles were found in the perinuclear space but not within the inner nuclear membrane; they were associated with ‘viroplasms’ and occurred commonly in membrane-bound vesicles. They apparently matured by budding from cytoplasmic membranes.
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Characterization of Vibriophage VA-1
More LessSUMMARYA bacteriophage isolated from the vaccine strain Vibrio cholerae NIH41 has been characterized by a variety of methods. The phage, called VA-1, is stable at 4 °C, sensitive to temperatures above 20 °C, stable at pH’s between 5 and 10, and more resistant to u.v. irradiation than most coliphages. The velocity constants for adsorption to V. cholerae RV79 and serum neutralization are 5.5 × 10−9 ml/min and 20, respectively. The latent period of VA-1 is about 45 min and its unusually large average burst size is 500 to 600.
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A Direct Estimate of the Number of Cowpea Chlorotic Mottle Virus Particles Absorbed by Tobacco Protoplasts that become Infected
More LessSUMMARYAt the most, 760 cowpea chlorotic mottle virus particles are sufficient to infect a tobacco protoplast.
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Induction of Interferon in Chick Cells by Polyoma Virus
More LessSUMMARYPolyoma virus induces high levels of interferon in non-permissive chick embryo fibroblast cultures, and certain features of this induction have been examined. In non-pre-treated cultures interferon first appears at around 30 h after infection and reaches maximum levels at around 70 h. An input multiplicity of at least 0.5 p.f.u./cell is required for induction. In cultures pre-treated with interferon (primed), interferon induction is enhanced, with interferon first appearing in the medium at around 20 h and reaching 4- to 8-fold higher levels than in non-primed cultures. In this case interferon is induced by an input multiplicity of as low as 0.1 p.f.u./cell. ‘Full’ or intact polyoma particles induce, but ‘empty’ shells do not. Interferon can also be induced by polyoma on continuously passaged lines of chick embryo fibroblasts.
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Interferon: the Dissociation of rIn.rCn-induced Proteins by Protonation
More LessSUMMARYMouse interferon, when induced by the synthetic ribopolymer, rIn.rCn, has a mol. wt. of about 150000. On brief exposure to low pH (3.5) conditions, the protein is dissociated into a smaller molecule (average mass of 38000 daltons); during this transition, the recoverable antiviral activity increases twofold. Our data support the idea that the high mol. wt. form may be a discreet interferon aggregate brought about by association of sub-units, although we cannot exclude the possibility that the antiviral protein (interferon) is simply adventitiously bound to a serum protein which antagonizes the antiviral action of interferon. This report provides additional support to the hypothesis, developed from studies with virus-induced mouse and human interferons, that apparently different interferon molecules may simply be different oligomeric species of a basic interferon subunit, α.
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Surface Antigen on Hamster Cells Transformed in vitro by two Strains of Rous Sarcoma Virus
More LessSUMMARYHamster cells transformed in vitro by the Bryan (RB 12) and the Schmidt-Ruppin (RS 2) strains of Rous sarcoma virus were studied for the presence, at their surface, of the virus-induced surface antigen (VISA). VISA expression, as detected by an in vitro colony-inhibition test, was higher in the RS 2 cells than in the RB 12 cells. Cytotoxic antisera directed against the two kinds of cells cross-reacted. Chicken cells infected by a non-transforming virus (RAV-1) did not elicit the appearance of detectable cytotoxic antibodies in hamsters.
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