- Volume 41, Issue 2, 1978
Volume 41, Issue 2, 1978
- Articles
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Electron Microscopy of Nicotiana clevelandii Leaf Cells Infected with Hypochoeris Mosaic Virus
More LessSUMMARYIn infected Nicotiana clevelandii mesophyll cells, the rod-shaped particles of hypochoeris mosaic virus were scattered throughout the cytoplasm, loosely aggregated in spherical cytoplasmic masses, present in regular arrays or associated with crystalline inclusions. The particles were 21·2 to 22·4 nm in diam. with a central canal approx. 5 nm in diam. Particles found in one membranebound aggregate were all 615 nm long. The crystalline inclusions (lattice spacing 9·8 nm) were membrane-enclosed, often rectangular in section and up to 2·3 × 1·5μm; the associated virus particles were found occasionally within the crystals, but more often between the enclosing membrane and the periphery of crystals.
The virus showed some similarities in its intracellular occurrence to four morphologically similar but serologically unrelated viruses (wheat soil-borne mosaic, potato mop-top, broad bean necrosis and beet necrotic yellow vein) and to some strains of tobacco mosaic virus, observations supporting its possible membership of the tobamovirus group.
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Genomic RNA of the Murine Coronavirus JHM
More LessSUMMARYGenomic RNA extracted from the purified murine coronavirus JHM sediments between 52S and 54S in aqueous sucrose gradients. The RNA is single-stranded and has an apparent mol. wt. of 5·4 to 6·5 ×106, as determined by electrophoresis in polyacrylamide agarose gels of different concentrations. The presence of polyadenylate sequences in the RNA is demonstrated by binding to oligo-(dT) cellulose and digestion with ribonucleases A and T1. The purified RNA does not dissociate into subunits at high temperatures or in high concentrations of DMSO and is infectious.
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The Effects of some Different Metabolic Inhibitors on Interferon Superinduction
More LessSUMMARYThree different inhibitors of RNA synthesis, actinomycin, α-amanitin and camptothecin, and five different inhibitors of protein synthesis were able to superinduce interferon production in human diploid fibroblasts treated with poly(rI). poly(rC).
Camptothecin was shown to be a reversible inhibitor of virus induced interferon formation. It also substantially reduced the interferon yield from human diploid fibroblasts which had been superinduced with actinomycin D and cycloheximide. This suggests that the previously reported failure of camptothecin to inhibit interferon production in human diploid cells after induction with poly(rI).poly(rC) is the result of two mutually opposing effects: a marked inhibition of interferon messenger RNA synthesis, but a stimulation of the activity of the interferon messenger RNA that is formed.
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Isolation and Characterization of Polysheaths, Phage Tail-like Defective Bacteriophages of Alcaligenes eutrophus H 16
More LessSUMMARYPolysheaths were spontaneously formed inside cells of the hydrogen bacterium Alcaligenes eutrophus H 16. These particles are long tube-like structures of 24 nm diam. belonging to the phage tail-like defective bacteriophages (Lotz, 1976). In mid log-phase fermenter-grown cells, polysheaths were observed in about 20 % of all cells sectioned. Evidence is provided for an inhibition of cell fission by polysheaths. Polysheaths were isolated by differential centrifugation and precipitation techniques using PEG and antibodies. The morphology of polysheaths was investigated electron microscopically by negative staining, ultrathin sectioning and metal shadowing. A surface lattice of the polysheath was derived from light optical diffraction data. The particles were also characterized by their biochemical and biophysical features: mol. wt. of the subunit determined by SDS-gel electrophoresis (58000), amino acid composition, isoelectric point (4·4), u.v. absorbance spectrum indicating the absence of nucleic acid, buoyant density (1·258), and stability against denaturants and proteolytic enzymes.
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Early Events in the Interaction Between Foot-and-Mouth Disease Virus and Primary Pig Kidney Cells
More LessSUMMARYFoot-and-mouth disease virus (FMDV) attached to pig kidney cells at 0 °C and could only be recovered in a form with a sedimentation coefficient and buoyant density lower than that of the native virus. Incubation of the virus-cell complex at 37 °C caused disruption of about 80% of the particles into a 12S protein sub-unit that had the same polypeptide composition as that produced by reducing the pH of the virus below pH 7. The remaining 20% had the same polypeptide and RNA composition as the native virus but it had a lower sedimentation coefficient, buoyant density and specific infectivity. These lower values are probably due to the association of the virus with cell membrane components. The 12S subunits were shown to be located inside the cell, indicating that disruption of the virus had occurred within the cell. The results are discussed in relation to the different cell mediated alteration of other picornaviruses.
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Phage Growth Characteristics on Stationary Phase Achromobacter Cells
More LessSUMMARYThe growth characteristics of α3a bacteriophage on stationary phase Achromobacter strain 14 are described. Phage α3a growth on stationary phase cells is characterized by a long and variable latent period of 6 to 9 h and an increased burst size of 710 p.f.u./cell as compared with 153 p.f.u./cell in exponential wild type cells. During the latent period the infected cells are very sensitive to changes in growth conditions and in particular, dilution. Pre-conditioning of the bacterial cells by allowing them to stand for 24 h after shaking for 3 days is an important aspect of the stationary phase phage growth system. Cells which have been allowed to stand retain the ability to be infected and to support phage growth for at least 16 days. Shaking cultures gradually lose the ability to support phage growth but the phage can persist in the host cell for 10 days until removal from shaking when the lytic cycle can proceed after allowing the cultures to stand.
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Photosensitization of Herpes Simplex Virus Type 1 with Neutral Red *
More LessSUMMARYCommercial neutral red (NR) originally containing at least 8 components was purified by thin layer chromatography. Herpes simplex virus type 1 (HSV-1) treated in vitro with 30 μg/ml of purified NR became sensitive to light inactivation within 2 min but rapidly lost this sensitivity upon dilution. Similarly, virus grown in the presence of NR lost its photosensitivity upon dilution of the virus stock. In both cases the kinetics of inactivation appeared to be multi-hit. Photoinactivation of intracellular virus was most effective when NR was applied between 6 and 12 h post-infection. The most efficient inactivation occurred when virus at pH 8·8 was irradiated by light at a wavelength of 470 nm.
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Host Antigen as the Sulphated Moiety of Influenza Virus Haemagglutinin
More LessSUMMARYInorganic sulphate (35S) was incorporated into the haemagglutinin molecule of A/Memphis/1/71 (H3N2) influenza virus when a keratosulphate-like host antigen was also incorporated into the glycoproteins of virus grown in the chorioallantoic membrane of the embryonated hen’s egg. Little or no 35S-sulphate was incorporated when this host antigen was not present in the glycoproteins of virus grown in chick embryo kidney cells or in the chorioallantoic membrane of embryonated duck eggs.
The presence of the keratosulphate-like host antigen was required for the stability of the haemagglutinin molecule in sodium dodecyl sulphate (SDS). The haemagglutinin molecules from virus grown in hens’ eggs were stable in SDS, whereas those from virus grown in duck eggs or in chick embryo kidney cells were not and could not be isolated on cellulose acetate.
Chemical analysis showed that there were 87 glucosamine residues and three molecules of sulphate per haemagglutinin subunit as calculated for a trimer molecule having a mol. wt. of 200000. There was one sulphate molecule per HA1 polypeptide chain and this was associated with the slowest migrating carbohydrate-protein complex of an HA1 tryptic digest separated by polyacrylamide gel electrophoresis.
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Selective Stimulation and Differentiation of Early Antigens in Lymphoblastoid Cell Lines Producing Epstein-Barr-Like Viruses
More LessSUMMARYComparisons of early antigens (EA) of EBV-related lymphotropic herpes virus of Old World primates have proved difficult to accomplish because antigen levels are low in infected cells and the ratio of virus capsid antigen to EA is generally high. To overcome these difficulties, we have developed a procedure which combines antigen stimulation with inhibition of late virus functions and results in the selective stimulation of EA. Using this procedure, we have examined the EA of EBV, herpesvirus papio, h. pongo, and h. pan. The results show that the EA of these viruses are composed of both R (restricted) and D (diffuse) components and that the EA, while related, are distinguishable.
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Isolation and Phenotypic Characterization of Human Adenovirus Type 2 Temperature-Sensitive Mutants
More LessSUMMARYThirty-nine temperature-sensitive (ts) mutants that fail to grow at 39·5 °C but develop normally at 33 °C have been isolated from a nitrous-acid-treated stock of a wild-type strain of type 2 human adenovirus. The frequency of ts mutants among the surviving viruses was about 10%. Complementation tests in doubly infected cell cultures at restrictive temperature permitted the assignment of 19 of these mutants to 11 complementation groups. They were characterized phenotypically according to their soluble capsid antigen production quantified by two-dimensional immunoelectrophoresis, virus DNA synthesis, as measured by alkaline sucrose gradient sedimentation of 34S DNA, and virion morphogenesis, as analysed by electron microscopy of cell sections. Two complementation groups were defective for DNA synthesis, four for soluble hexon production and two groups for total penton (penton base + fibre), while one group revealed no fibre production. Two complementation groups presented a normal antigen pattern, but the particles exhibited altered morphology as observed in cell sections.
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Mastomys natalensis Papilloma Virus (MnPV), the Causative Agent of Epithelial Proliferations: Characterization of the Virus Particle
More LessSUMMARYA virus (MnPV) with the structural characteristics of papilloma viruses was isolated from benign and malignant proliferations of adult animals of the inbred line ‘GRA Giessen’ of Mastomys natalensis. The particles can be banded in CsCl gradients at densities of 1·34 g/ml (full particles) and 1·29 g/ml (empty particles). The virus DNA has a buoyant density of 1·7104 g/ml and can exist in three different conformations (supercoiled circular, nicked circular and linear), the sedimentation values of which have been determined as 23 to 24S, 16 to 17S and 14 to 15S, respectively. Although the mol. wt. of MnPV DNA is similar to that of HPV 1 DNA, the size of the fragments obtained after cleavage of MnPV DNA with the restriction endonuclease Hae III is quite different from the pattern seen with human papilloma virus. The virion contains 12 different polypeptides; the major structural protein has a mol. wt. of 56000. MnPV is shown to be the causative agent of the skin proliferations, because tumours can be induced by inoculation of purified virus, whereas no cutaneous alterations are observed when the particles are inoculated in the presence of anti-MnPV serum. MnPV can be re-isolated from the experimentally induced tumours.
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Efficient Transfer of Interferon-Induced Virus Resistance between Human Cells
More LessSUMMARYThe rate of development of interferon-induced virus resistance in a mixture of two human cell types (U and WISH) is determined by the cell type (WISH) in the mixture which responds first. This phenomenon has been shown with two types of interferon assay procedure, and with both vesicular stomatitis virus and Sindbis virus. The transfer of virus resistance from one human cell (WISH) to another (U) (homospecific transfer) is much more efficient than the transfer from mouse L cells to WISH cells (heterospecific transfer), as shown by a much lower ratio of donor to recipient cells required for maximum transfer as well as a more rapid transfer. Thus, virus protection afforded by the interferon system is amplified more efficiently in mixtures of different human cells than in mixtures of mouse and human cells. These results suggest that, in a mixed population of cells such as occurs in vivo, more slowly responding cells might be influenced by cells which respond more rapidly to interferon. A defensive role is suggested for this mechanism which amplifies protection due to interferon.
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The Isolation of Recombinants between Related Orbiviruses
More LessSUMMARYTemperature-sensitive mutants of the related orbiviruses, Wallal and Mudjinbarry, recombine with high frequency when grown in pairs in cell culture. The genome of each virus consists of discrete segments of double-stranded RNA and high frequency recombination suggests that reassortment of genome segments occurs rather than classical recombination. Electrophoresis in acrylamide gels of RNA extracted from the progeny of a cross between Wallal ts 101 and Mudjinbarry ts 3 mutants, revealed that three plaque isolates of 60 tested differed in RNA pattern from each of the parent viruses and from each other. Further analysis of the electrophoretic profiles suggested that the isolates were recombinants with RNA segments derived from each of the parent viruses.
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Virus Mutation during ‘Slow Infection’: Temporal Development and Characterization of Mutants of Visna Virus recovered from Sheep
More LessSUMMARYVisna virus could be recovered from peripheral blood leukocytes of sheep for years after intracerebral inoculation. Viruses recovered from sheep prior to and several months after development of antibody were antigenically identical to the parental strain used for inoculation. Subsequently, mutant viruses which were not neutralized by the animals’ sera were obtained. Longitudinal studies of leukocyte viruses collected from two infected sheep showed that more than one strain of virus could co-exist in the animal. Virus neutralization tests using sequentially collected sera and the viruses recovered from leukocytes revealed a sequential development of antibody to parental and then to each strain of mutant virus. Characterization of two of the mutant viruses showed that they were antigenically stable, virulent in cell culture and when inoculated into new sheep, elicited antibodies which cross reacted with the parental virus from which they were derived. This continuous mutation of Visna virus in persistently infected sheep may be a mechanism for the production of chronic disease.
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Assembly of Semliki Forest Virus Nucleocapsid: Detection of a Precursor in Infected Cells
More LessSUMMARYThe synthesis of Semliki Forest virus nucleocapsid in infected cells was studied by labelling the virus RNAs with 3H-uridine for different periods at various phases of infection. Short pulses (10 to 20 min) revealed the accumulation of 42S RNA in a ribonucleoprotein which sedimented at about 90S (90S RNP) and contained only small amounts of capsid protein. Only after longer pulses was the labelled 42S RNA found in the virus nucleocapsid, suggesting that the 90S RNP may be its precursor. The life time of the 90S RNP was long in the early phases of infection and short in the late phases, reflecting the increased rate of assembly of the nucleocapsid during infection. The 90S RNP was the only 42S RNA containing RNP found in cells infected with temperature sensitive mutants deficient in nucleocapsid formation or wild type infected cells treated with cycloheximide to inhibit nucleocapsid assembly.
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Somatic 0–1 Antigen Conversion of Salmonella typhimurium by a Type B Phage P221dis, Hybrid between P22 and Fels 1 Phages
More LessSUMMARYA type B Salmonella phage P221, derived from recombination between a type A phage P22 and a type B phage Fels 1, carries the protein coat of Fels 1 and the P22 early genes, at least the c to h21 genes. One of the P221 strains, P221dis, is dismune over P221 lysogens and co-immune with P22. Thus it carries the Im gene (the second immunity region) of P22 to establish co-immunity with P22. Since the att region and a1 gene for somatic 0–1 antigen conversion of P22 are located between the Im and c genes, the P221dis prophage attachment site and 0–1 antigen of P221dis lysogens were analysed. P221dis prophage is integrated at the attP22 site near the pro A region of the bacterial chromosomes and expresses the somatic 0–1 antigen.
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An Improved Method for Determining Neutralizing Antibody against Lymphocytic Choriomeningitis Virus in Human Sera
More LessSUMMARYHuman antibody neutralizing lymphocytic choriomeningitis virus is most reliably determined in mice as assay hosts. Whereas the previously recommended procedure yielding a neutralization index requires much serum, the method described here uses only 0·1 ml. An equal mixture of virus and serum is incubated and residual infectivity is titrated intracerebrally in mice. The neutralizing activity is given by the ratio of virus surviving after incubation with control serum and with the test serum respectively. This ratio is called the neutralization factor.
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Reversion to Virulence of Attenuated Canine Distemper Virus In Vivo and In Vitro
More LessSUMMARYReversion to virulence of dog kidney cell attenuated canine distemper virus (CDV), Rockborn strain, was demonstrated after serial passage in dogs. The same strain also reverted to virulence after serial passage in canine macrophage cultures. Criteria for virulence in dogs included clinical signs, weight loss, elevated body temperatures, reduced total lymphocyte counts, impaired blastogenesis responses of blood lymphocytes, appearance of virus antigen in epithelial cells, the character of neutralizing antibody responses and the capability of re-isolated virus to grow in canine macrophage cultures.
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A Re-appraisal of the Biochemical Map of Foot-and-mouth Disease Virus RNA
More LessSUMMARYThe proteins induced by infection of BHK 21 cells with foot-and-mouth disease virus have been compared by tryptic peptide analysis. The results indicate that there are three primary products 5′–P88, P52, P100–3′. The polypeptide P56, which we considered previously to be a primary product, is derived from the region of the genome that codes for P100. The results indicate that there are alternative cleavage pathways of P100, the polypeptide coded for by the 3′ end of the genome.
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DNA Sequences in Influenza Virions
More LessSUMMARYDuring the propagation of A (H3N2) influenza virus in chick embryos, incorporation of 3H-thymidine into virions takes place, whereas no such incorporation occurs with Newcastle disease virus. Incorporation of 3H-thymidine is a result of DNA synthesis. This virion-associated DNA is present in cores obtained after treatment of virions with bromelain.
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Effect of Interferon on the Cell Cycle of BALB/c 3T3 Cells
More LessSUMMARYThe effect of interferon on the exponential growth phase of BALB/c 3T3 cells was studied. Although interferon reduced the growth rate and the proportion of cells in both the M and S phases (mitotic index and labelling index), there were no appreciable differences in the duration of these phases between control and interferon-treated cells. Moreover, the shape of the first peak of the fraction of labelled mitoses (FLM) curve was not altered by treatment with interferon, which indicates that the duration of the S and G2 phases was not affected. However, the height of the second peak of the curve in interferon-treated cells was extremely reduced as compared to control culture. These results are compatible with the idea that the suppressive effect of interferon is exerted mainly in the G1 phase (A-state) of the cell cycle of BALB/c 3T3 cells.
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Ultrastructural Study of Virus-like Particles in Chinese Hamster Lung Cells
More LessSUMMARYVirus-like particles were found in the E36 cells. One type, associated with centrioles, consisted of two concentric shells with a diam. of 50 to 60 nm. Some of these particles were seen budding through the plasma membrane giving rise to a free immature particle showing two concentric shells and an outer envelope. Concentration of the inner shell into a nucleoid results in a mature particle characterized by a nucleoid, mainly eccentrically located, surrounded by an intermediate layer and wrapped in an envelope. The diameter of the mature and immature particles was 75 to 85 nm. The morphogenesis of this virus-like particle resembles that of the oncoviruses.
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Effect of Interferon on Transcription and Translation of Vesicular Stomatitis Virus in Human and Simian Cell Cultures
More LessSUMMARYThe effect of interferons on vesicular stomatitis virus (VSV) primary transcription, amplified RNA synthesis [i.e. the sum of primary transcription RNA replication (leading to [–] RNA) and secondary transcription (leading to [+] RNA) and virus protein synthesis were studied. In a human cell line, both human and simian interferons inhibited the initiation of primary transcription and amplified RNA synthesis. In contrast, in a simian cell line tested similarly, the initiation of these activities was not affected, though they decreased as the infection progressed. Nevertheless, virus protein synthesis was completely inhibited. These results demonstrate that the action of interferon on virus transcription and/or translation may depend more on the host cell than on the particular interferon used.
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Synthesis of Herpes Simplex Virus DNA in Preparations of Chromatin from Infected Cell Nuclei
More LessSummaryChromatin prepared from cells infected with Herpes simplex virus type 1 or type 2 can synthesize both virus and cell DNA in vitro. The rate of synthesis is comparable to that of isolated whole nuclei. Incorporation is limited, and both cell and virus DNA synthesis are sensitive to the presence of virus-specific antiserum and phosphonoacetate. In chromatin from cells infected with a phosphonoacetate resistant virus mutant, both types of DNA synthesis are resistant to the presence of the inhibitor.
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Characterization of Adenovirus Type 12 Tumour Antigen Produced in Chick Fibroblasts
More LessSUMMARYTumour (T) antigen was characterized in non-permissive chick fibroblasts and permissive HEp-2 cells infected with Ad12 either in the presence or in the absence of cytosine arabinoside. Antiserum against T antigen specifically immunoprecipitated two polypeptides of apparent mol. wt. 50000 and 11000.
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Effect of Trypsin Treatment on the Antigenic Characteristics of Plaque Variants of Type O1 and Type Asia-1 Foot-and-Mouth Disease Viruses
More LessSummaryAntigenic differences were demonstrated between the large and small plaque variants of both types O1 and Asia-1 foot-and-mouth disease viruses. Treatment of the large and small plaque variants of the viruses with trypsin essentially abolished the observed antigenic differences. Thus, these plaque variants have antigenically different trypsin-sensitive determinants that may influence their immunogenicity and infection capabilities.
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Presence of a Covalently Linked Protein on Calicivirus RNA
More LessSummaryThe infective RNA of the calicivirus, vesicular exanthema virus, has been shown to contain a protein which is apparently linked to the RNA by a covalent bond. The protein remained bound to the RNA after boiling with SDS-mercaptoethanol-urea or treating with formamide-dimethylsulphoxide but was removed by incubating with proteinase K. The mol. wt. of the protein was estimated to be about 10 × 103 by electrophoresis in highly cross-linked polyacrylamide gels. The infectivity of the RNA was destroyed by removal of the protein with proteinase K.
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