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Volume 5,
Issue 4,
1969
Volume 5, Issue 4, 1969
- Articles
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Interferon and Resistance to the Toxic Effects of Influenza Virus in vivo
More LessSUMMARYWhen mice were inoculated intravenously with Newcastle disease virus, interferon was formed, and the mice were protected against the lethal effects of a subsequent intravenous injection of a toxic strain of influenza A virus. Mice were similarly protected when injected intravenously with a suspension of mouse macrophages which had been stimulated to produce interferon by treatment in vitro with Newcastle disease virus. It is known that the reticuloendothelial system has a role in the production of interferon and in the development of non-specific resistance to virus infections. When this system was blocked completely by a thorotrast injection, or partially as the result of splenectomy, there was a sharp decrease both in the amounts of interferon formed in mice in response to an injection of Newcastle disease virus and in their resistance to the toxic effects of influenza virus.
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Inapparent Persistent Virus Infection in Continuously Grown Aëdes aegypti Mosquito Cells
More LessSUMMARYLong-term inapparent infections were established in continuously grown uncloned Aëdes aegypti mosquito cells inoculated with Semliki Forest virus and incubated at 28°. This response of mosquito cells to infection, which contrasts with the outcome of infection by the same virus of mammalian cells at 37°, was due to a small proportion of virus-producing cells in the culture, and a low yield of virus per infected cell.
The percentage of virus-producing cells was between 2.8 and 8% at the height of infection, and between 0.01 and 0.09% at the lowest level of infection. The yield of virus per infected cell was estimated to be between 1 and 6 p.f.u.
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Growth of Vesicular Stomatitis Virus in a Continuous Culture line of Antheraea eucalypti Moth Cells
More LessSUMMARYVesicular stomatitis virus was grown in a tissue culture line of Antheraea moth cells and examined by plaque assay in mammalian cells, immunofluorescence and electron microscopy. After infection the virus content of Antheraea cells increased for 2 to 4 days and then remained relatively constant. There was no obvious cytopathogenic effect on the host cells. Immunofluorescence showed that only a fraction of the cell population was producing a significant amount of virus antigen. This fraction was dependent on the m.o.i. and was equal to
when the m.o.i. was 50 p.f.u./cell. At this multiplicity electron microscopy of cell sections revealed the presence of large quantities of virus associated with some of the cells. Budding of vesicular stomatitis virus from the moth cell membrane was also confirmed by electron microscopy.
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Electron Microscope Study of Human NB and SMH Cells Infected with the Parvovirus, H-1: Involvement of the Nucleolus *
More LessSUMMARYThe sequence of changes in human NB and SMH cells that occurred after H-1 virus infection, was studied with the electron microscope. The earliest alterations in NB cells after infection were detected in the nucleolus, after 31 hr. The pars fibrosa appeared devoid of its formed elements and was occupied instead with mainly ‘incomplete’ H-1 virus. The pars granulosa was more diffuse than normal and its nucleolar granules more sharply defined. A few ‘incomplete’ and ‘complete’ virus particles were scattered about the nucleus at approximately the same time. The cytoplasm was intact. Shortly thereafter, margination of the nuclear chromatin occurred. The nucleoli became increasingly condensed and shrunken, and apparently formed a doughnut-shaped body with condensed walls containing empty virus. Eventually, most of the nucleolar elements disappeared as the nuclei filled with complete and incomplete virus particles as well as occasional crystals possibly of protein nature. As the nucleolus disappeared, the cytoplasm disintegrated except for a few scattered mitochondria and some recognizable areas of endoplasmic reticulum which contained complete virus particles in linear array. Seventy-seven hr after infection when the nucleus had also broken down, virus was found associated with the disintegrated nucleus, within fragments of endoplasmic reticulum, and attached to thickened and adjoining plasma membranes of the few remaining, apparently uninfected cells.
In SMH cells basically similar changes occurred except that the nucleoli appeared to fragment 31 hr after infection. Later, though most of the nucleolar sections disappeared, a few persisted and formed ‘nucleolar inclusions’ identical to those observed in NB cells. In contrast to the mixture of ‘complete’ and ‘incomplete’ virus observed in NB cells, the virus found in SMH cells was almost exclusively ‘empty’. Virus seen outside the nucleus, either in NB or SMH cells, appeared complete.
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Adsorption of Mycobacteriophage on Cell-wall Components
T. Imaeda and F. San BlasSUMMARYHeat-killed whole cells, purified cell walls, and the three main polymers isolated from cell walls of Mycobacterium sp. atcc 607, M. smegmatis atcc 14468 and M. phlei atcc 11758, were examined to find the receptor sites for phage GS-7. Among heat-killed whole cells of insusceptible species, M. smegmatis adsorbed the phage at a higher rate than the susceptible Mycobacterium sp. atcc 607, whereas M. phlei did not adsorb the phage at all, suggesting a distinction between phage-resistance (absence of receptor) and phage-immunity (presence of both receptor and repressor).
Each of the three polymers of the cell wall (the lipopolysaccharide of the outer layer, the lipopolysaccharide-lipoprotein complex of the middle layer and the lipopolysaccharide-mucopeptide or mucopeptide complex of the inner layer) contain receptor substances. The lipopolysaccharide moiety isolated from the lipopolysaccharide-lipoprotein complex adsorbed phage at the same rate as the whole complex; implying that the possible receptor substances were the lipopolysaccharide moieties of the three polymers. Delipidation of both cell wall and the lipopolysaccharide-mucopeptide complex resulted in a loss of phage-adsorbing capacity, suggesting that the lipid portion of the lipopolysaccharides may constitute the phage receptors.
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Proteins Specified by Influenza Virus in Infected Cells: Analysis by Polyacrylamide Gel Electrophoresis of Antigens Not Present in the Virus Particle
More LessSUMMARYFive polypeptides were found when purified influenza virus was analysed by electrophoresis on polyacrylamide gel. Previous work has shown that hyperimmune convalescent antibody prepared in mice and adsorbed with purified influenza virus which had been disrupted with ether still gave precipitin lines in immunodiffusion tests with extracts of cells infected with influenza virus. These presumed non-structural antigens, labelled with radioactive amino acids, were precipitated with the virus-adsorbed antibody preparation. A comparison of the precipitate and purified virus by polyacrylamide gel electrophoresis revealed three polypeptides which differed from the polypeptides of the virion. These were probably virus specific non-structural proteins.
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The Host Range of bryan Strain Rous Sarcoma Virus Synthesized in the Absence of Helper Virus
More LessSUMMARYDifferent L-R ‘non-producer’ cell lines synthesized one of two types of RSV(O), distinguishable by host range. RSVα(O) has no known natural host. RSVβ(O) is infectious for Japanese quails, European pheasants and some Brown Leghorn, White Leghorn and Reaseheath I × C hybrid chickens. The host range of RSVβ(O) is distinct from other avian tumour viruses. Host susceptibility to RSVβ(O) is genetically determined in a complex and as yet unclear way. It is not correlated with response to the A, B, and C sub-groups of avian tumour viruses or with presence of the natural antigen which reacts in the COFAL test: nor is it sex-linked. RSVβ(O) is not markedly oncogenic in hamsters.
The difference between RSVα(O) and RSVβ(O) did not appear to be a heritable property of the RSV particle but depended on the type of host cell in which the virus was cloned. Synthesis of RSVβ(O) occurred only after passage through COFAL positive host cells. The possible control of the infectious properties of RSVβ(O) by a helper virus present in an elusive form in permissive hosts is discussed.
A stock of inactivated Sendai virus commonly used for cell fusion was apparently contaminated with an active avian leukosis virus.
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Interference and Neutralization Studies with Bryan Strain Rous Sarcoma Virus Synthesized in the Absence of Helper Virus
More LessSUMMARYThe relationship of RSVβ(O) to other avian tumour viruses was studied by virus interference and serological cross neutralization. Viruses of subgroup B interfere with RSVβ(O) on chicken cells but not on quail cells. Viruses of sub-groups A and C enhance the focus forming efficiency of RSVβ(O). Antisera to RSVβ(O) and avian tumour viruses of sub-group A, B and C do not cross-react, but serological cross-reaction was found between RSVβ(O) and sub-group D.
The ability of RSV(O) and L-R cells to stimulate anti-viral antibodies has been investigated.
No helper virus in RSVβ(O) stocks was demonstrable by interference assays.
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Paradoxical Effect of Freund’s Complete Adjuvant upon Transplantation Efficiency of Adenovirus-induced Tumour Cells
More LessAdjuvants appear to act by creating a depot from which antigen is slowly released and by stimulating antibody-forming cells (Ehrich et al. 1945). Freund’s adjuvant consists of an emulsion of water in paraffin oil stabilized by lanolin derivatives (Freund, Casalas-Ariet & Genghof, 1940). Its action is potentiated by adding dead mycobacteria or mycobacterial lipid (complete adjuvant) and Freund attributes the cellular response to the mycobacterial substance (Freund, 1956). Freund’s complete adjuvant (FCA) can produce delayed hypersensitivity of extreme severity to a foreign antigen and at times sensitize an animal to its own tissue (Freund, Thompson & Lipton, 1955) (auto-immunity). Transplantation and tumour immunity are chiefly of the cellular type (delayed hypersensitivity) and FCA appears to be an ideal agent to enhance such immunity. However, we observed increased tumour growth when hamsters were treated with FCA before challenge with adenovirus tumour cells. The present report describes this paradoxical enhancing effect.
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Plaque Formation by an Arbovirus in a Mosquito Cell Line
More LessEstablished lines of insect cells have been obtained only recently and their use in arbovirus studies is only beginning (Suitor, 1966; Converse & Nagle, 1967; Rehacek, 1968; Singh & Paul, 1968 a, b; Banerjee & Singh, 1968; Yunker & Cory, 1968). Most arbovirus growth reported in insect cell lines does not cause concomitant cytopathic effect (CPE) and plaque formation has not been reported before.
The cell line employed in this study originated from tissues of the mosquito Aëdes albopictus (Singh, 1967). The cells were cultured in a manner identical to that described by Singh (1967) and complete monolayers were obtained about 5 days after subculture. Because this line contains cell types of various sizes, a single cloning was made in order to isolate the smallest cell type. Small coverslips were placed in Petri dishes and dilutions of cell suspensions added. After incubation, coverslips containing single colonies were transferred to tubes in order to increase the cell population.
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The Growth Accelerating Effect of Bittner Virus in Monolayers of Baby Mouse Kidney Cells
More LessMurine mammary tumour virus (Bittner virus) is generally detected and titrated by in vivo tests. The time required for these tests varies from about 4 months to more than 1 year according to the clinical criteria accepted (Nandi, 1963). Recently Charney, Pullinger & Moore (1968) reduced this time to about 3 months by inoculation of newborn female mice and the detection of Bittner virus antigens in the milk of young adults by immunodiffusion tests. The immunological technique alone does not discriminate between infective and non-infective Bittner virus antigens. A faster in vitro assay would speed up the research of many aspects of the virus, including those at the cellular and molecular levels. Biological effects of Bittner virus preparations in vitro have been described, but an in vitro assay does not yet exist.
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A Capsule-depolymerizing Enzyme from Alcaligenes faecalis Infected with Bacteriophage A6
I. J. Mare and J. A. SmitPhage-associated enzymes which act on capsules of hosts with the production of plaques surrounded by halos have been reported for a number of capsulated Gramnegative bacteria (Bartell, Orr & Lam, 1966; Chakrabarty, Niblack & Gunsalus, 1967). Organisms in the halos are devoid of capsules but are viable and in some instances become sensitive to the action of different phages. This suggests that hidden phage receptors may have been exposed (Sutherland & Wilkinson, 1965). This communication describes the isolation and action of a capsule-depolymerizing enzyme associated with an Alcaligenes faecalis phage host system.
Alcaligenes faecalis bacteriophage a6 produces plaques with depressed haloes on the capsulated host A. faecalis strain a6 (Mare, de Klerk & Prozesky, 1966). Haloes are not produced on an uncapsulated spontaneous variant a6t of strain a6. The haloproducing agent is a capsule-destroying enzyme.
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Protein Synthesis in BHK 21 Cells Infected with Foot-and-Mouth Disease Virus
More LessMacromolecular synthesis in mammalian cells following infection with picorna-viruses has been studied by several groups (Baltimore & Franklin, 1962; Penman et al. 1963; Plagemann, 1968). However, much of the evidence is contradictory and does not lead to a unified hypothesis accounting for the observed effects. This was demonstrated clearly by the work of Plagemann (1968) in which infection of L cells with mengovirus resulted in a rapid inhibition of cellular RNA and protein synthesis, whereas neither process was affected when Novikoff rat hepatoma cells were used. The confusion in this area of biochemistry was reviewed clearly by Martin & Kerr (1968).
Our previous studies (Brown, Martin & Underwood, 1966) indicated that infection of BHK 21 cells with foot-and-mouth disease virus (type SAT 1) inhibited cell protein synthesis within 15 to 30 min. after infection.
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Enhancement of Encephalomyocarditis Plaques by Saponin
More LessDuring the titration of encephalomyocarditis virus on primary mouse cells it was observed that both the size and number of plaques were increased by pretreatment of the cells with saponin which was being used to lyse erythrocytes.
Crude brown saponin was freshly dissolved for each experiment to give a 2% solution in 50% ethanol. Saponin from batch 205470/630707 prepared by British Drug Houses Ltd was used for all experiments. Cells were obtained by trypsinization (Dulbecco, 1952) of the embryos from 17-day pregnant Porton white mice. Before centrifugation the trypsinized cells were exposed for 5 min. at room temperature to 0.02% saponin. They were then centrifuged and grown as monolayers in 10% calf serum in medium 199. The cultures were infected with Habel’s large plaque (τ) mutant of encephalomyocarditis virus grown in mouse brain and were incubated in 5% CO2 under an overlay of medium 199 containing 0.176% bicarbonate, 5% calf serum, 5% chick embryo extract and 0.75% Difco Noble agar.
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Presence of Unusual Virus Particles in Two Hamster Tumour Tissue Culture Cell Lines Induced by Murine Sarcoma Virus
More LessA murine sarcoma virus (MSV-h) which causes sarcomas and other lesions in mice, rats, Praomys and hamsters was described by Harvey (1964, 1968), Chesterman et al. (1966) and East & Harvey (1968). Mouse or rat tumours induced by MSV-h and grown in tissue culture readily released virus infective for both species; in contrast virus produced by hamster tumour cultures was active only in hamsters (Bassin et al. 1968; Harvey, 1968). One such hamster tumour culture, B34 established by Dr R. Bassin, is currently being maintained in our laboratory. ‘Hamster specific’ MSV-h, when examined by electron microscopy, cannot be distinguished from fully infective MSV-h or any of the well known type C murine leukaemia viruses (Chesterman et al. 1966; Bassin et al. 1968).
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A Difference in the Buoyant Density of Hæmagglutinin From Rubella Virus Strains
More LessThe multiplication of rubella virus in tissue culture cells results in the production of two complement fixing antigens (Schmidt et al. 1968), two antigens detectable by immunodiffusion (Le Bouvier, 1969), an antigen detectable by haemagglutination and infective virions heterogeneous with respect to buoyant density (Magnusson & Skaaret, 1967). The physical characteristics of rubella virus haemagglutinin have been studied by equilibrium density gradient centrifugation in caesium chloride and values of 12.5 (Palmer & Murphy, 1968) and 1.33 (Amstey, Hobbins & Parkman, 1968) have been obtained. Strain differences in buoyant density have been noted for herpes simplex virus (Roizman & Roane, 1961). As part of a continuing study of rubella strains (Oxford, 1969), we have determined the buoyant density in caesium chloride of the haemagglutinin prepared from four virus strains with widely varying numbers of tissue culture passes.
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