- Volume 27, Issue 3, 1975
Volume 27, Issue 3, 1975
- Articles
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Replication of Reticuloendotheliosis Viruses in Cell Culture: Chronic Infection
More LessSUMMARYAfter an initial acute infection with cell killing, chicken or duck embryo fibroblasts infected in culture with reticuloendotheliosis viruses set up a chronic infection with no cell killing or morphological transformation. Essentially all of the chronically infected cells produced virus. The virus production was not sensitive to cytosine arabinoside or mitomycin C as was virus production in an acute infection.
The chronically infected cells had a strong group-specific resistance to the c.p.e. of superinfecting reticuloendotheliosis viruses. However, they were sensitive to vesicular stomatitis virus and avian leukosis-sarcoma viruses.
After double infection, single cells produced reticuloendotheliosis virus and avian leukosis-sarcoma virus.
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Penicillium stoloniferum Virus: Altered Replication in Ultraviolet-derived Mutants
More LessSUMMARYPhenotypic mutants of the wild type of Penicillium stoloniferum NRRL 5267 were obtained from conidia exposed to ultraviolet light for 60 min (10% survival). Virus content of the wild type and of nine phenotypic mutants was determined by polyacrylamide gel electrophoresis. Four mutants had no detectable Penicillium stoloniferum virus F (PsV-F), whereas the other five had levels of PsV-F in the mycelium similar to the wild-type strain. All nine mutants and the wild type had comparable levels of Penicillium stoloniferum virus S (PsV-S). Maximum virus levels occurred after 9 days of submerged culture in a 2% yeast extract-15% sucrose medium. Virus replication in the fungal host continued after protein, RNA and DNA synthesis levelled off. Virus levels ranged from 85 to 150 E 260 units (extinction units at 260 nm in 1 cm cell) per 4.7 to 5.3 g dry weight of mycelium for the mutant strains compared to 106 E 260 units per 4.2 g dry weight of the wild-type strain.
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The Proteins of Murray Valley Encephalitis Virus
More LessSUMMARYProteins specified by Murray Valley encephalitis virus were labelled during virus growth in Vero and in PS cells, and separated by polyacrylamide gel electrophoresis. The purified virus particle contains three proteins (V-1, V-2 and V-3) whereas the slow sedimenting haemagglutinin or virus sub-particle lacks the core protein V-2 but contains NV-2, a non-structural protein. Seven non-structural proteins in addition to V-2 and V-3 were identified in infected cells. Electrophoretic profiles of virus-specified proteins in both cell lines were almost identical after elimination by a double-label technique of the background of continuing host-cell protein synthesis. Glucosamine was incorporated into the envelope protein V-3 and NV-2. From 26 to 46 h post-infection in Vero cells, the proportion and amounts of virus-specified proteins remained constant and they were non-equimolar; incorporation of labelled leucine into V-2 was much greater than incorporation into NV-2, whereas in cells infected with Kunjin (a related flavivirus) this ratio of incorporation was reversed. At 21 to 25 h, the synthesis of V-2 was less prominent but there was an enhanced synthesis of NV-X. Apart from V-1, NV-1, NV-4 and NV-5, all the proteins are larger than the corresponding Kunjin virus proteins and together represent about 400 × 103 daltons of polypeptide synthesis, which is close to the maximum coding content of the flavivirus genome.
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Tobacco Rattle Virus in Tobacco Mesophyll Protoplasts: Infection and Virus Multiplication
More LessSUMMARYPoly-L-ornithine greatly increased infection of protoplasts by tobacco rattle virus (TRV), and had the largest effect when incubated with virus particles for at least 10 min before inoculation. Using a final concentration of 1 μg/ml TRV particles and 1 to 1.5 μg/ml poly-L-ornithine in 0.025 M-phosphate buffer, pH 6.0, to inoculate mesophyll protoplasts of tobacco cv. Xanthi by the ‘indirect’ method, up to 98% of the intact protoplasts became infected. When the protoplasts were stored overnight at 5 °C before inoculation, 95% became infected. In protoplasts kept at 22 °C after inoculation, about half the yield of infective particles was produced during the first day and almost all the remainder during the second. The final yield was about 2 × 105 long virus particles and 6 × 105 short particles per infected protoplast. Fluorescent antibody staining showed that TRV coat protein antigen accumulated throughout the cytoplasm. In electron micrographs, the long TRV particles were associated with mitochondria whereas the short particles were generally dispersed in the cytoplasm. Infective RNA was produced after inoculation with long particles but TRV coat protein antigen, and long and short TRV particles, were made only in protoplasts inoculated with both kinds of particle; infection was not detected in protoplasts inoculated with short particles alone.
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Electron Microscopical Observations of the Structure of the Virus of Viral Haemorrhagic Septicaemia (VHS) of Rainbow Trout (Salmo gairdneri)
More LessSUMMARYNegative staining of particles of the Danish F1 strain of viral haemorrhagic septicaemia virus grown in rainbow trout gonad-2 cells revealed three different types of particles: bacilliform, bullet-shaped, and long particles. The average size of the first two types was 165 × 65 nm. The long particles had a length of up to 3000 nm and showed a close resemblance to Marburg virus. Well defined surface projections could be found in all particle types. Without any special treatment we were able to demonstrate different disintegration stages of VHS virus particles in all preparations.
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A Morphological Study of the Internal Component of Influenza Virus
More LessSUMMARYRapid treatment of influenza virus directly on the microscope grid with non-ionic detergent has allowed better visualization of the internal component. Many micrographs show that this ribonucleoprotein (RNP) is present as a continuous strand of 6 nm diam. arranged in the form of a double coil or helix. In spite of the minimal treatment to which the virus was subjected most helices still showed signs of degradation. The findings that we have obtained lead us to suggest that the RNP component of influenza virus must be very sensitive to both chemical and physical manipulations, any of which could cause it to fracture from one continuous strand into several pieces, although such breakages could possibly occur at specific points along its length.
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Protein Synthesis in Sendai Virus-infected Cells
More LessSUMMARYThe rate of protein synthesis in chicken embryo cells infected with Sendai virus 18 to 20 h previously was about two times greater than in mock-infected controls. At this time of infection six stable virus-induced proteins, four major structural proteins (P, NH, NP and M) and two non-structural proteins (28K and 61K), were identified by electrophoresis in SDS-polyacrylamide gel of total cell extracts. The structural glycopeptide F was not detected in the infected cell extracts. Pulse-chase experiments showed that P, NP, M and 28K proteins either did not undergo any post-translational processing or the processing occurred very rapidly. By contrast, a glycopeptide NH was apparently derived from one of two unstable precursors, 69K or 63K, which were revealed only after a short pulse. The synthesis of virus-specific proteins appeared to be regulated since its rate varied for individual classes of proteins.
In nucleocapsid-like particles isolated from infected cells two major structural proteins (P and NP) were found. A minor component with a very large mol. wt. was revealed in these particles as well as in the virus particle.
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Early Alteration of Poliovirus in Infected Cells and Its Specific Inhibition
More LessSUMMARYHeLa cells infected with radioactive poliovirus type 2 were disrupted with ultrasonic treatment, followed by addition of a non-ionic detergent. Two types of virus particles were found to sediment at 80 to 90% the rate of native virus. The first of these appeared to be a complex of native virus particles and membrane components, since treatment with 0.2% SDS released infectious native particles. The second was non-infectious and its sedimentation rate was not greatly altered by SDS. One hour after infection this non-infectious particle was the major product of cell-mediated eclipse.
We have confirmed that 10 to 30 μg/ml S-7, a substituted thiopyrimidine, blocks infection of cells by poliovirus in a specific manner. Analysis of cells infected with radioactive poliovirus type 2 in the presence of S-7 showed that the virus particles remained as the complex which can be disrupted with SDS. In addition to blocking cell-mediated eclipse, S-7 stabilizes poliovirus against heat inactivation in vitro at the same concentrations which block infection. This action resembles the effect of 10−2 M-glutathione, which is also known to block cell-mediated eclipse of poliovirus.
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Characterization of Adenovirus Antibodies by Single Radial Diffusion in Agarose Gels Containing Immobilized Intact Virus Particles
More LessSUMMARYThe interaction between antibodies and surface antigens of intact immobilized adenovirus particles was studied by single radial immunodiffusion tests (SRDT) in agarose gels. Purified virus particles and antisera against virus particles of selected serotypes representing different subgroups and against structural components of certain serotypes were employed. Antibodies against hexons, pentons and fibres all gave visible zones. Antisera against the latter components were more efficient in forming zones and this was thought to be due to the occurrence of relatively smaller amounts of vertex capsomeres and fibre antigen at the virus particle surface. The capacity of antibodies against vertex capsomeres to form zones was demonstrated in tests with virus particles of intermediate types.
The SRDT with immobilized virus particles was found to be highly sensitive for the demonstration of virus particle surface antigens shared between serotypes. Results obtained in previous studies using virus particle haemagglutination-inhibition (HI) tests including anti-antiserum were confirmed. Cross-reactions mainly occurred within subgroups and were ascribed to a sharing of vertex capsomere antigens. In preliminary studies a rapid typing was achieved by examination of paired human sera from five cases of adenovirus infections.
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A Genetic Recombination Map of Foot-and-Mouth Disease Virus
More LessSUMMARYSixty ts mutants were isolated from the Pacheco strain of type O foot-and-mouth disease virus after treatment with either 5-fluorouracil or hydroxylamine. The conditions affecting recombination and assay of the ts + recombinants were standardized. Using two ts mutants resistant to guanidine, three-factor crosses, supported by two-factor crosses, located 34 of the mutations in a linear arrangement. The recombination frequencies between certain pairs of mutations were additive. The guanidine character of the two resistant mutants mapped as a single site mutation and was located near the middle of the map.
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Physico-chemical and Serological Characterization of Five Rhabdoviruses Infecting Fish
More LessSUMMARYViruses isolated from fish with viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), spring viraemia of carp (SVC), swim-bladder inflammation (SBI) and pike fry disease (PFD) have been grown to high titre in fathead minnow cells. While our preparations of the IHN, SVC, SBI and PFD viruses showed typical rhabdovirus morphology with bullet-shaped particles and distinct surface projections, the VHS virus preparations had a less typical rhabdovirus morphology but were pleomorphic with a preponderance of flexuous rods. Using virus labelled with [3H]-uridine, it was shown that each virus contained RNA which sedimented at 38 to 40 S and was hydrolysed by very low concentrations of ribonuclease. The viruses of SVC, PFD and SBI had a polypeptide composition similar to that of vesicular stomatitis virus, the prototype rhabdovirus, but the IHN and VHS viruses gave a pattern similar to that of rabies virus. In serum neutralization tests the SVC and SBI viruses were indistinguishable. VHS virus showed no serological relationship with the other four viruses but there was a low level of cross-reaction between the PFD, IHN and SVC-SBI viruses.
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Isolation and Characterization of a Rod-shaped Bacteriocin from a Strain of Rhizobium
L. Gissmann and W. LotzSUMMARYA bactericidal agent (‘bacteriocin 16-2’) produced by rhizobial strain 16-2 has been characterized as a sheathless rod-shaped particle with a length of 200 nm and a diam. of 8 nm. One end of the rod is pointed and carries short fibre-like appendages, while the other end appears square. The particles specifically adsorb with their pointed end to bacteriocin-sensitive, but not to bacteriocin-resistant, cells. The possible mode of action of this bacteriocin is discussed.
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Chromatographic Studies on Picornavirus Capsid Polypeptides
More LessSUMMARYThe polypeptides of encephalomyocarditis, Mouse-Elberfeld and type 5 rhinoviruses behave similarly when chromatographed on calcium phosphate (brushite), each being eluted by a linear phosphate buffer gradient containing sodium dodecyl sulphate in three major peaks, C1, C2 and C3. Analysis of the peaks by polyacrylamide gel electrophoresis suggests that the major capsid polypeptides of these three picornaviruses elute in the order: δ (peak C1), γ with β (peak C2) and α (peak C3).
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Modifications of Cellular RNA-polymerase II after Infection with Frog Virus 3
More LessSUMMARYRNA-polymerase II extracted from FV3-infected and uninfected BHK cells were compared by measuring their abilities to bind [3H]-amanitin and ribonucleoside triphosphates. Binding sites for [3H]-amanitin and the dissociation constant of the complex between [3H]-amanitin and RNA-polymerase II were significantly modified following FV3 infection. The apparent Km’s for ribonucleoside triphosphates remained unchanged.
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Efficacy of Exogenous Interferon Treatment Initiated After Onset of Multiplication of Vesicular Stomatitis Virus in the Brains of Mice
More LessSUMMARYInitiation of treatment with potent interferon preparations (6.4 × 106 units per dose) 4 days after intranasal inoculation of VSV (at a time when virus has already multiplied in the brains of most mice) resulted in a marked increase in mouse survival. In a series of 6 experiments only 11 to 18% of control mice survived whereas 52% of interferon treated mice survived. These results suggest the usefulness of exogenous interferon even when treatment is begun late in the course of an acute virus disease.
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Neuraminidase Content of Strains of Newcastle Disease Virus which Differ in Virulence
More LessSUMMARYThe neuraminidase content of purified virus particles of three velogenic strains of Newcastle disease virus was compared with that of three lentogenic strains. Velogenic strains possessed approximately three times as much enzyme as lentogenic strains.
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The Lack of Antiviral Effect of (Polyinosinic Acid): (Polycytidylic Acid) when Attached to Insoluble Supports
More LessSUMMARYA local antiviral effect can be observed when (poly rI).(poly rC), bound to Visking discs by u.v. irradiation, is incubated with monolayers of human foreskin fibroblast cells. Radioactive labelling of cytosine residues in (poly rI).(poly rC) with 125I, has provided a much more sensitive method for determining the fate of the insoluble (poly rI).(poly rC) than has been available hitherto. The antiviral effect is not related to the amount of (poly rI).(poly rC) present on the insoluble support but rather to the amount of polynucleotide lost from the support during incubation. Treatment of (poly rI).(poly rC) which had been bound to cyanogen bromide-activated Sepharose with either dilute alkali or pancreatic ribonuclease released virtually all the polynucleotide. A small amount of (poly rI).(polyrC) is released from the insoluble matrix in the presence of serum-free Minimum Eagle ’s Medium.
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