- Volume 42, Issue 2, 1979
Volume 42, Issue 2, 1979
- Articles
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Purification and Some Properties of Two Viruses Infecting the Cultivated Mushroom Agaricus bisporus
More LessSUMMARYHighly purified preparations of the isometric mushroom viruses 1 and 4 were made by a combination of differential centrifugation, ion-exchange chromatography on DEAE-cellulose and sucrose density-gradient centrifugation. Purified preparations of MV1 virions (25 nm diam.) sedimented at 130S contained one capsid polypeptide of mol. wt. 24400 and two species of double-stranded RNA of mol. wt. approx. 1.4 × 106. MV4 particles (35 nm diam.) had a sedimentation value of 140 to 145S, contained one capsid polypeptide of mol. wt. 63800 and two species of dsRNA of mol. wt. 1.5 × 106 and 1.4 × 106. MV1 and MV4 were antigenically unrelated.
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Isolation and Biological Properties of Drosophila X Virus
More LessSUMMARYDrosophila X virus described here appeared as a contaminant in Drosophila melanogaster. It is pathogenic for the inoculated flies, inducing anoxia sensitivity and death in these insects. An assay based on these symptoms in flies has been developed. Immunofluorescence has been used to study the characteristics of infected Drosophila cell cultures. A permanent infection can be established in these cultures. This virus is morphologically similar to several ungrouped vertebrate and invertebrate viruses like IPNV, IBDV and Tellina tenuis virus. Its possible origin is discussed.
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Decreased Production of Transforming Virus and Altered Antigenic Behaviour in Cultured Avian Sarcoma Cells
More LessSUMMARYTumours induced in chickens by inoculation of avian sarcoma viruses are frequently capable of undergoing spontaneous regression. It is only those tumour cells which have been derived from progressively growing neoplasms that are able to produce transforming progeny virus in vitro and to shed into the culture medium antigens which are specifically reactive with the peripheral lymphocytes of sarcoma-bearing hosts. Following multiple passages and extended growth in culture, however, the ability of these tumour cell fluids to stimulate the lymphocytes of sensitized hosts diminishes in concert with the declining capacity of these cells to continue to synthesize fully transforming progeny virus. In certain instances, however, aged tumour cells are able to synthesize particles which contain the enzyme RNA-dependent DNA polymerase yet lack detectable envelope glycoprotein.
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Stimulation of Ornithine Decarboxylase by Human Cytomegalovirus
More LessSUMMARYHuman cytomegalovirus (HCMV) infection of low serum-arrested confluent whole human embryo (Flow 5000) cells markedly stimulated ornithine decarboxylase (ODC) activity. Increased ODC activity was apparent by 12 h post-infection. The capacity of HCMV to stimulate ODC was: (1) dependent upon multiplicity of infection; (2) eliminated when the virus was neutralized with specific antiserum; and (3) sensitive to ultraviolet irradiation. Virus-mediated induction, in contrast to high serum induction of ODC, was not subject to inhibition by polyamines added to the growth medium.
Phosphonoacetic acid (PAA) which blocks HCMV replication by inhibiting the activity of HCMV-specific DNA polymerase and which does not prevent HCMV-induced stimulation of cell DNA synthesis, reversibly inhibited HCMV-induced stimulation of ODC activity by 74%. Studies with PAA indicated that HCMV-induced stimulation of ODC activity is independent of cell DNA synthesis and that the mechanism regulating virus-induced stimulation may be related to the HCMV-specific DNA polymerase.
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The Neutralization of Transmissible Gastroenteritis Virus by Normal Heterotypic Serum
More LessSUMMARYExposure of transmissible gastroenteritis virus of pigs to unheated normal heterotypic serum resulted in a drop in infectivity, an effect which was lost after heating the serum to 56 °C for 30 min or by treatments inactivating complement. Analysis of virus proteins, RNA and lipids, and centrifugation studies showed little difference between inactivated and control virus, but electron microscopy of negatively stained particles after treatment with serum revealed holes in the virus envelope, characteristic of those caused by complement in the presence of antibody.
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Establishment and Characterization of Indian Muntjak Cell Lines Transformed with Simian Virus 40
More LessSUMMARYKidney cells of an Indian muntjak were transformed with simian virus 40 (SV40). The transformation efficiency of the tertiary cultures was very high when estimated by the agar suspension culture method. The efficiency was about 0.015% when infected at an input multiplicity of 0.4 p.f.u./cell. Clonal cell lines were established from the colonies in soft agar medium. Most of the cell lines and their subclones produced a small amount of infectious SV40. The SV40 virion antigen-positive cells in a clone increased from 0.2% to about 40% by the treatment with mitomycin C. More than 70% of the cells in two cell lines were normal in G- and C-banded karyotypes, indicating that chromosomal change is not a necessary step in the process of transformation of the Indian muntjak cells with SV40.
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Enhancement of Infectivity of Encephalomyocarditis Virus RNA by Amphotericin B Methyl Ester
More LessSUMMARYThe methyl ester of amphotericin B (AmBME), a macrolide polyene antibiotic, enhanced the infectivity of encephalomyocarditis (EMC) virus RNA for L929 cells. AmBME alone (100 µg/ml) resulted in increases in EMC virus RNA infectivity of 10- to 100-fold. Addition of DEAE dextran at concentrations (5 µg/ml), which alone slightly suppressed EMC virus RNA infectivity, further augmented the effects of AmBME (augmentation in infectivity up to 750-fold). AmBME did not inhibit RNase, did not enhance EMC virus infectivity and increased infectivity of EMC virus RNA which was already cell-associated. The polyenes are probably acting by increasing intracellular penetration of polyribonucleotides.
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Effect of Calcium Ions on the Infection of Bacillus subtilis by Bacteriophage SF 6
More LessSUMMARYInfection of Bacillus subtilis 168Wt by SF 6 resulted in a rapid reduction in the number of phages. This could be counteracted by the addition of calcium, barium or strontium ions. At the optimum concentration of 7.5 × 10−2 m, the number of p.f.u. remained constant until lysis began. Although cultures of another host, B. subtilis 31 try− his−, at the end of the logarithmic growth phase produced a substance which inactivated free phages, this was not the major cause of the reduction in the numbers of p.f.u. during infection experiments at low Ca2+ concentrations. The diminution of the number of p.f.u. was therefore attributed to the fact that at least one of the steps of the lytic cycle was calcium dependent. Adsorption of SF 6 was equally effective in media containing high or low concentrations of calcium ions. Infection experiments with phages whose DNA had been labelled radioactively revealed that, at high concentrations of calcium ions, the label remained associated with the host cells until lysis commenced. At low concentrations, however, a dissociation between phage DNA and the host was found, although adsorption took place at a normal rate. From these experiments we concluded that a high concentration of calcium ions was required for the penetration of phage DNA. Similar experiments with phages whose protein coat had been labelled showed the same results, indicating that desorption of the inactivated phages occurred. Both electron microscopy and column chromatography with hydroxyapatite showed that a considerable fraction of the inactivated phages had ejected their DNA into the medium. A hypothesis explaining these results is presented.
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Demonstration by Electron Microscopy of Intracellular Virus in Acholeplasma laidlawii Infected with either MV-L3 or a Similar but Serologically Distinct Virus (BN1 virus)
More LessSUMMARYCultures of Acholeplasma laidlawii strain M1305/68 were inoculated with Mycoplasmatales virus-laidlawii 3 (MV-L3) and examined by electron microscopy. Particles resembling MV-L3 were observed both intra- and extracellularly in thin sections prepared from MV-L3 infected cultures, but not from uninfected cultures. Similar particles were occasionally observed in uninoculated cultures of A. laidlawii strain BN1 cells, from which a virus (BN1 virus) was subsequently isolated. This virus was morphologically similar but not identical to MV-L3. It also differed serologically from, and in its resistance to, MV-L3 and the other mycoplasma viruses.
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Synthesis of Proteins in Tobacco Protoplasts Infected with Brome Mosaic Virus
More LessSUMMARYThe proteins synthesized in protoplasts infected with strain V5 of brome mosaic virus have been studied. Four new proteins of mol.wt. 2, 3.5, 10 and 10.7 × 104 were observed. These account for over 90% of the virus genome. The three proteins with lowest mol.wt. corresponded closely in size to those observed in protoplasts infected with cowpea chlorotic mottle virus.
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Rift Valley Fever Virus: Some Ultrastructural Observations on Material from the Outbreak in Egypt 1977
More LessSUMMARYRift Valley fever virus isolates from the 1977 outbreak in Egypt were studied at an ultrastructural level. The particles measured 90 to 110 nm in diam. using negative staining and sectioning techniques, with a core component of 80 to 85 nm. The surface of the virions was calculated to be covered by approx. 160 sub-units. The particles were found in smooth endoplasmic reticular systems, which were made up of either multi-tubular complexes, or of a single large vacuole. The majority of these membrane systems were found to be unassociated with Golgi apparatus. Inclusion bodies were found within the host cell nuclei (made up of rods and fine granules) and in the cytoplasm (aggregates of fine or coarse granules). The possible relationship of these structures to virus replication is discussed.
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Transformation of Rat Cells by the Hybrid Virus Ad22+ HEY
More LessSUMMARYA set of four isogenic rat cell lines transformed by Ad22+ HEY have been studied. While all of the cell lines synthesize SV40 T antigen, only one expresses adenovirus 2 T antigen: none expresses SV40 V antigen or adenovirus 2 fibre antigen. Three cell lines contain 1 to 2 virus equivalents of SV40 and adenoviral sequences per diploid quantity of rat cell DNA and the fourth line contains five copies of SV40 and 20 copies of the adenovirus genome. At least three of the cell lines contain DNA sequences from the helper adenovirus 2 in addition to sequences from the Ad2+ HEY genome. The patterns of integrated virus sequences are complex suggesting multiple insertions of both adenovirus and SV40 DNA sequences. SV40 can be rescued from three cell lines by fusion with permissive cells.
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Cross-Neutralization Study of Seven California Group (Bunyaviridae) Strains in Homoiothermous (PS) and Poikilothermous (XTC-2) Vertebrate Cells
More LessSUMMARYAntigenic relationships among seven California group strains were studied by a plaque-reduction neutralization test (PRNT). Cross-reactions occurred in most cases but three subgroups were noted: (1) the major serogroup contained the viruses of California encephalitis, LaCrosse, Snowshoe Hare and Ṫrahyňa (including the Lumbo strain) whereas (2) Jamestown Canyon and (3) Trivittatus viruses were distinct. There was no significant difference between the PRNT results in mammalian (PS) cells incubated at 37 °C and amphibian (XTC-2) cells incubated at 28 °C. Trivittatus virus failed to produce plaques in XTC-2 cells.
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Mechanisms of Interferon Induced Transfer of Viral Resistance Between Animal Cells
More LessSUMMARYThe sequence of events initiated by interferon and leading to the antiviral state were studied as possible sites for the cell-to-cell transfer of interferon induced viral resistance. The possible role of interferon produced by recipient cells was negated by the demonstration of transfer of resistance in the presence of anti-human interferon antibody and under conditions of a single cycle of VSV growth. Transfer of sensitivity of WISH cells to mouse interferon, possibly through transfer of a membrane receptor, seems unlikely since resistance was transferred in the absence of mouse interferon. From kinetic data and the fact that actinomycin D blocked resistance in human cells for 3 h longer than in mouse cells, it seems unlikely that the mouse antiviral protein itself or its mRNA alone is a likely candidate for the transfer of resistance. Thus, by a process of elimination, we suggest that secondary messenger molecules which transmit the interferon signal from the membrane to the nucleus are the effector substance(s) for the transfer process.
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Variant Lines of Mouse Kidney Cells Transformed by an SV40tsA Mutant with Growth Properties of Wild-Type Transformed Cells at Nonpermissive Temperature
H. Otsuka, D. R. Dubbs and S. KitSUMMARYmKSA207 cells, a BALB/c mouse kidney line transformed by a tsA mutant of SV40, are temperature-dependent for the expression of the ‘standard transformed phenotype’. At the permissive temperature (33.5 °C), the mKSA207 cells resembled wild-type (wt) SV40 transformants; they contained the intranuclear SV40 T antigen, grew to high saturation density in monolayer culture in either 10% or 0.5% serum, and also in methylcellulose suspension culture and became multinucleate in cytochalasin B. At the nonpermissive temperature (39.8 °C), the mKSA207 cells lost some of their transformed properties; they grew only to low density in 10% serum, hardly grew at all in 0.5% serum or in methylcellulose suspension culture, and remained mono- or binucleate in cytochalasin B. At 40 °C in low serum, mKSA207 cells lost the intranuclear T antigen and when fed 10% serum at 39.8 °C, accumulated large amounts of T antigen in the cytoplasm. Derivatives of mKSA207 have been selected at 39.8 °C in liquid medium and methylcellulose suspension culture. The heat-adapted lines, like wt SV40 transformants, exhibited the standard transformed phenotype at both 33.5 and 39.8 °C. It is unlikely that acquisition of temperature-independence for the transformed phenotype was due to reversion of the tsA gene to wild-type because the heat-adapted cell lines displayed the cytoplasmic T antigen at 39.8 °C, characteristic of the parental mKSA207 cells and SV40 rescued from one of the heat-adapted lines was temperature sensitive for growth. The T antigen levels (complement fixation units per 106 cells) of heat-adapted lines grown at 39.8 °C were comparable to those of mKSA207 cells grown at 33.5 or 39.8 °C.
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Infection of Tobacco Mesophyll Protoplasts with Tobacco Necrotic Dwarf Virus, a Phloem-limited Virus
S. Kubo and Y. TakanamiSUMMARYTobacco mesophyll protoplasts were successfully inoculated with tobacco necrotic dwarf virus (TNDV), a virus closely related to potato leafroll virus. The optimum conditions for infection as assessed by staining with fluorescent antibody to virus particles were 1 µg/ml TNDV, 1 µg/ml poly-l-ornithine, 0.025 m-phosphate buffer, pH 5.4, and 1 to 2 × 105/ml protoplasts: this combination resulted in 70 to 80% infection. Accumulation of virus antigen was first detected 12 h after inoculation, and both the number of fluorescing protoplasts and intensity of fluorescence increased markedly during the following 20 h. Virus antigen was distributed throughout the cytoplasm. Electron microscopy of thin sections revealed groups of virus-like particles in the cytoplasm and a few similar particles in the nucleus. Virus yield was estimated to be 7.8 × 105 particles per infected protoplast at 30 h after inoculation. Accumulation of virus antigen was completely inhibited by cycloheximide, but was not affected by actinomycin D or chloramphenicol, when they were applied 2 h after inoculation. Studies on the replication of aphidborne, phloem-limited viruses should be greatly facilitated by using the mesophyll protoplast system.
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Type C Virus Induced by Iododeoxyuridine in the Human Embryonic Cell Strain HEL-12
More LessSUMMARYThe induction of a type C virus from a strain of human embryonic lung cells (HEL-12) by iododeoxyuridine (IdUrd) was examined at various times during in vitro propagation. IdUrd did not elicit type C virus production immediately following initiation of cultures from frozen primary HEL-12 cells. After overnight treatment with 30 µg/ml IdUrd the cells expressed viral antigens and produced a type C virus between the 25th and 80th day of in vitro growth. Production of the induced type C virus was transient. Single-cell clones of the parental HEL-12 strain were likewise susceptible to type C virus production by IdUrd. The ability of IdUrd to induce virus terminated with the onset of spontaneous type C virus production from HEL-12 cells at between 80 and 120 days of in vitro growth.
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Differentiation Between SV40 Large-T and U Antigenic Sites
More LessSUMMARYRadioimmune precipitation, SDS-polyacrylamide slab gel electrophoresis and fluorography were used to investigate the SV40 large-T and U antigenic sites on species of proteins synthesized during wild type and tsA58 mutant infections in TC7 monkey cells. Wild type infection at 33 and 41.5 °C and the A58 infection at 33 °C produced similar profiles of three species ranging in mol. wt. from 84000 to 94000, all of which had both the large-T and U antigenic sites. The A58 infection at 41.5 °C, however, produced an additional four discrete species ranging in mol. wt. from 60000 to 74000 that contained the large-T site(s), but not the U site(s). A subpopulation of the 74000 mol. wt. species contained both sites. Therefore, the region of the A58 mutant 94000 mol. wt. species containing the U antigenic site(s), the COOH-terminal region, appears to be more sensitive to processing, probably proteolytic cleavage, than does the region containing the large-T antigenic site(s).
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SV40-Transformed Human Diploid Cells that Remain Transformed throughout Their Limited Lifespan
More LessSUMMARYOf ninety-one subcultured foci of SV40-transformed WI-38 human diploid fibroblasts, none yielded cells that grew indefinitely, but all cells in each subculture continued to produce T antigen and to look morphologically transformed throughout their lifespan. These results are consistent with the commitment theory of fibroblast senescence, but predict that a special transformation event is necessary to account for the rare survivors.
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