- Volume 53, Issue 2, 1981
Volume 53, Issue 2, 1981
- Announcement
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- Bacterial
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Achromobacter sp. 2 Phage α3: A Physical Characterization
More LessSUMMARYPhage α3 had a buoyant density of 1.506 g/ml, suggesting a protein capsid. The mol. wt. of the phage genome was 34 ± 1 × 106. A dual genome population was observed and partial denaturation, duplex and restriction analyses facilitated distinction between constituent phages, α3τ and α3ω. Phage α3τ exhibited a 0.7 ± 0.2 × 106 mol. wt. deletion in its genome with respect to α3ω. Both phages were encapsulated by a headful mechanism and the genomes were permuted over 27% and <;18% of their length respectively. It is concluded that stable variants may be isolated and that these phages offer potential, through physical studies, to elucidate the properties of the Achromobacter-α3 system.
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The Morphology of Staphylococcal Bacteriophage K and DNA Metabolism in Infected Staphylococcus aureus
P. J. Rees and B. A. FrySUMMARYThe morphology and dimensions of bacteriophage K particles were determined by electron microscopy. This virus had an icosahedral head (approx. 70 nm diam.) and a long (210 nm) thin (15 nm) contractile tail which terminated in a complex basal appendage. The precise dimensions of the particles were dependent on the negative stain employed. The buoyant densities of the K virus particle and its DNA were 1.479 g/ml and 1.689 g/ml respectively. The DNA had a base composition of 30% G + C, a contour length of 16.1 µm and a calculated mol. wt. of 33 × 106. With Staphylococcus aureus (NCTC 9318) as host, the latent period was 25 min, the eclipse period 14 min and the average burst size 60 p.f.u./bacterium. Infection resulted in inhibition of host DNA synthesis and degradation of the bacterial DNA: the products were used for the synthesis of phage DNA. The kinetics of DNA synthesis in infected and uninfected bacteria were examined. There was no initial cessation of DNA synthesis in the infected bacteria.
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- Animal
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Analysis of Genome Composition and Reactogenicity of Recombinants of Cold-adapted and Virulent Virus Strains
More LessSUMMARYTemperature-sensitive (ts) mutations occurring in three genes 1, 2 and 7, and 1, 5 and 7 were found in two cold-adapted (ts) attenuated influenza virus strains A/Leningrad/9/37/46 (H0N1) and A/Leningrad/134/17/57 (H2N2) respectively. The recombinants, obtained by crossing these cold-adapted strains with virulent influenza virus strains, had different genome structures and inherited from one to six genes from the cold-adapted parents. Tests of reactogenicity of recombinants in volunteers showed all the recombinants to be non-reactogenic irrespective of the number of genes inherited from the cold-adapted parent.
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Adenylic Acid: Deoxythymidine 5′-Phosphotransferase: Evidence for the Existence of a Novel Herpes Simplex Virus-induced Enzyme
More LessSUMMARYBHK (dPyK−) cells infected with herpes simplex virus type 1 (HSV-1) contain a virus-induced deoxythymidine (dThd)-phosphorylating enzyme. This enzyme uses AMP as phosphate donor and is called AMP:deoxythymidine 5′-phosphotransferase (or kinase). The enzyme was purified over 1300-fold and was found to be specific for an AMP substrate. It can thus be distinguished from virus-specific deoxypyrimidine kinase (dPyK). It was shown that the two substrates AMP and dThd participate in the reaction at a 1:1 molar ratio; the K m for AMP was 2.3 µm and for dThd it was 2.1 µm. The mol. wt. of the enzyme was estimated to be between 110000 (by glycerol gradient centrifugation) and 90000 (by gel filtration). For optimum activity, the phosphotransferase required an alkaline pH, and 37 °C; the activation energy of the reaction was 18450 cal/mol. The appearance of the enzyme after infection parallels that of viral DNA synthesis-related functions.
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Production and Screening of Cell Hybrids Producing a Monoclonal Antibody to Human Interferon-α
More LessSUMMARYMice and rats were immunized with human lymphoblastoid interferon, a mixture of human leukocyte (interferon-α) and human fibroblast (interferon-β) interferons, and their serum anti-interferon levels measured. Anti-interferon activity was detected in all animals, although the mouse sera had higher titres than the rat sera. The spleens of two mice were removed and used to prepare hybrid myelomas. Anti-interferon activity in tissue culture supernatants was measured using a direct neutralization assay and a clone (NK2) producing antibody to human interferon-α (leukocyte) was isolated. The anti-interferon activity of this monoclonal antibody was confirmed using a new indirect immunoprecipitation (IIP) assay, which shows some advantages over the direct neutralization assay. The antibody did not neutralize human interferon-β (fibroblast) or mouse interferon.
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The Distribution of Human Coronavirus Strain 229E on the Surface of Human Diploid Cells
More LessSUMMARYThe distribution of human coronavirus strain 229E (HCV 229E) particles on the surface of human diploid (MRCc) cells was examined. Virus particles showed a totally random distribution on fixed cells and on cells to which virus had been adsorbed in the cold. A marked redistribution of virus particles was observed on warming virus—cell preparations to 33 °C for 20 min, the peripheral areas of the cell becoming relatively devoid of virus particles while the majority of particles were now located some distance from the edge of the cell. Redistribution did not occur in the presence of metabolic inhibitors.
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Comparative Oligonucleotide Analysis of Exogenous and Endogenous Primate Type C Viruses
More LessSUMMARYRNAs of representative viruses of the exogenous simian sarcoma virus-gibbon ape lymphosarcoma virus (SiSV/GALV) and endogenous baboon virus (BaEV) classes of subhuman primate type C viruses were compared and related to HEL-12 virus, an isolate derived from human embryonic lung cells. The extent of sequence identity between different viral RNA preparations was determined by comparison of fingerprint patterns obtained after electrophoretic separation of RNase T1-resistant oligonucleotides. The studies presented indicate that HEL-12 viral RNA and simian sarcoma-simian associated virus [SiSV(SSAV)] RNA share 90 to 95% of the large oligonucleotides. From 5 to 10% of virus-specific oligonucleotides were detected in each of several virus preparations examined and their occurrence was independent of the cell line on which the virus was propagated. HEL-12 virus and GALV-SF have 50% unique oligonucleotides in common. These are the same oligonucleotides that are shared between GALV-SF and SiSV(SSAV) RNA. Two BaEV isolates, M7 and BILN, and RD114, a BaEV-related endogenous virus of cats, each easily display distinguishable oligonucleotide patterns. Large oligonucleotides characteristic for these three endogenous virus isolates were not detected in the fingerprints of HEL-12 virus, SiSV(SSAV) and GALV-SF.
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Alterations in Peptide Structure of Vesicular Stomatitis Virus Mutant and its Central Nervous System Isolate
More LessSUMMARYGradient SDS-polyacrylamide gel electrophoresis (PAGE) and proteolytic digestions were utilized to examine the virion proteins of two isolates of wild-type vesicular stomatitis virus (WT-VSV), WTATCC from the American Type Culture Collection and WTGL from Glasgow, as well as temperature-sensitive (ts) mutant ts G31 and a central nervous system (CNS) isolate of ts G31 designated ts G31BP. The WTATCC M protein differed in electrophoretic mobility and in its tryptic or chymotryptic peptide maps from the 125I-labelled M proteins in WTGL, ts G31 or ts G31BP. The M protein in the latter three viruses appeared identical using either tryptic or chymotryptic digestion procedures; however, limited digestion with V8 protease revealed a difference between the M protein of ts G31 and both WTGL and ts G31BP M proteins. The L, NS and G proteins all had identical tryptic and chymotryptic peptide maps in WTGL, ts G31 and ts G31BP virions. The N protein, however, was demonstrated to be distinctly different in the WTGL virion when compared with the ts G31 (or ts G31BP) virion by its tryptic peptide map. In addition, limited proteolytic digestion of the 125I-labelled N proteins revealed a different peptide structure in ts G31BP compared to N proteins of ts G31 or WTGL. The altered N protein in the CNS isolate, ts G31BP, is discussed in terms of its altered in vivo phenotype of labile viral RNA, and its potential role in the unique CNS disease associated with this virus.
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A Sensitive Method for the Detection and Isolation of Recombinants of Foot-and-Mouth Disease Virus
More LessSUMMARYRecombination between temperature-sensitive (ts) mutants of foot-and-mouth disease (FMD) virus was examined, using an infectious centre technique that was more sensitive (approx. 30-fold) than the conventional virus yield test. The test involved a brief incubation of the mixedly infected cells at the permissive temperature to allow recombination to occur followed by assay at the restrictive temperature to select for those cells in which recombination had occurred. With crosses involving widely separated mutations, as many as 28% of the infected cells produced presumptive recombinant plaques. Since each plaque was the result of an independent event, large numbers of different presumptive recombinants could be isolated for further study. Analysis of presumptive recombinant plaques from a variety of crosses showed that, in general, the virus produced had the properties expected of recombinants. An approximate correlation was found between genetic distance, as determined in the yield recombination test, and the percentage of recombinant infectious centres observed. The phenomenon was very sensitive to the balance between the input multiplicities of the two parent viruses and occurred very early in virus replication. The test has considerable potential for the study of genetic interactions in FMD virus, but it would be surprising if this potential was limited to picornaviruses.
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Interference Induced in GL-V3 Monkey Kidney Cells by Rabies Virus Strains
More LessSUMMARYResistance to superinfection with vesicular stomatitis virus (VSV) occurred in GL-V3 monkey kidney cells infected with the CVS-11, Pitman Moore, LEP Flury, but not the ERA strain of rabies virus. Specific immunofluorescent staining of intracellular rabies antigen showed that the number and size of fluorescent foci increased after the onset of interference, and that this was paralleled by increasing yields of infectious virus. Although CVS-11 and ERA differed in their ability to induce interference, the virus yields from monolayers infected with either strain were similar. Interference apparently had no effect on the replication or dissemination of the inducing virus, and seems unrelated to the long incubation period or aberrant forms of infection in vivo.
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Poly(A) Polymerase Activity in Human Rotavirus
More LessSUMMARYA poly(A) polymerase enzymic activity was found in partially purified preparations of a human rotavirus. The activity was demonstrated using conditions similar to those utilized for the detection of a poly(A) polymerase previously described in reovirus (incubation at 43 °C in 70 mm-tris buffer pH 7.5 containing 12 mm.Mn2+). The enzymic activity was associated only with complete, double-shelled particles. Characterization of the poly(A)-containing product of the in vitro reaction by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose in the presence of 7 m-urea, showed that it is composed of oligonucleotides of a chain length similar to, or slightly larger than, those observed in reovirus.
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Protein Synthesis in Cells Infected with Bovine Rotavirus
V. Urquidi, E. Novo and J. EsparzaSUMMARYBovine rotavirus was found to multiply efficiently in LLC-MK2 cells, a continuous line of rhesus monkey kidney, with a growth cycle which was essentially completed within 9 h after infection. The presence of low concentrations of trypsin (10 µg/ml) in the virus inoculum was essential for infectivity. Polyacrylamide gel electrophoresis of infected cell extracts demonstrated the synthesis of at least eight virus-specific polypeptides 6 h post-infection with mol. wt. ranging from 102 × 103 to 29 × 103. Six polypeptides (about p102K, p91K, p84K, p45K, p37K and p34K) were identified as structural components of the virion. Two other polypeptides (p54K and p29K) were identified as non-structural components. The synthesis of non-structural polypeptides appeared to precede that of the structural proteins. Pulse-chase experiments showed only one minor post-translation modification of the virus-specified proteins, namely an increase in the mobility of the 29K polypeptide.
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Antigenic Differences between the Membrane Antigen Polypeptides Determined by Different EB Virus Isolates
More LessSUMMARYThe antigenic specificities of the membrane antigen (MA) complex on three Epstein—Barr (EB) virus-producing cell lines have been compared by complete absorption of an anti-MA antiserum with P3HR-1 cells, followed by testing for residual anti-MA antibody activity against B95-8 and QIMR-WIL cells. Indirect membrane immunofluorescence showed that the absorbed serum, which had lost the capacity to bind P3HR-1 cells, nevertheless gave bright staining on a small proportion of B95-8 cells. SDS-polyacrylamide gel electrophoretic analysis of 125I-labelled MA polypeptides showed that the absorbed serum was able to isolate MA polypeptides from both B95-8 and QIMR-WIL cells, but not from P3HR-1 cells. The results demonstrate that there is substantial sharing of antigenic specificities between the MA determined by three isolates of EB virus, and some antigenic divergence.
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Absence of Activated Murine Leukaemia Virus in X-irradiated CBA/H-T6Crc Mice
More LessSUMMARYCBA/H-T6Crc mice, a substrain that does not normally express demonstrable levels of murine leukaemia virus (MuLV) and has a low natural incidence of leukaemia, were examined for evidence of virus activation at various times following X-irradiation. Although X-irradiation caused a high incidence of leukaemia, no ecotropic, xenotropic or recombinant MuLV was detected by in vitro co-cultivation of bone marrow, spleen and thymus cells from pre-leukaemic and leukaemic animals with selectively permissive cell lines followed by indirect immunofluorescence for MuLV group-specific (gs) antigen. These results, therefore, are not consistent with the hypothesis that endogenous viruses are the universal aetiological agents of leukaemia.
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Interferon-mediated Inhibition of Mouse Mammary Tumour Virus (Type B) and Mouse Leukaemia Virus (Type C) in the Same Culture
S. K. Arya and B. GordonSUMMARYThe sensitivity of mouse mammary tumour virus (MMTV) and murine leukaemia virus (MuLV) to interferon was tested by investigating the effect of interferon in cultures of mouse mammary tumour cells simultaneously synthesizing the two viruses. The dose—response curves showed that the extracellular production of both viruses by these cells was equally inhibited by interferon treatment. In contrast, the intracellular steady-state concentration of virus-specific RNA of both MMTV and MuLV was not significantly affected in these cells. This was the case regardless of whether total cellular RNA or polysomal poly(A)-containing RNA was examined. These results are consistent with the suggestion that interferon-caused inhibition is related to a host-mediated effect on a post-transcriptional step in virus synthesis.
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Interferon-Albumin Conjugate with Conserved Biological Activity
More LessSUMMARYThe heterobifunctional reagent N-succinilimidyl 3-(2-pyridylthio)propionate (SPDP) was used for the preparation of a disulphide-linked conjugate between Namalwa lymphoblastoid interferon and serum albumin. The linkage of interferon to albumin did not reduce its ability to protect MDBK cells against infection by vesicular stomatitis virus (VSV). The conjugate did not dissociate into free interferon and albumin under conditions prevailing in the assay for interferon activity. The rate of clearance of the interferon-albumin conjugate from the mouse circulatory system was somewhat slower than that of free interferon.
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- Plant
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Isolation and Characterization of Subviral Structures from Tomato Spotted Wilt Virus
More LessSUMMARYParticles of a stable isolate of tomato spotted wilt virus (TSWV) had a density of 1.147 g/ml in sucrose and contained four major structural proteins of mol. wt.: 1, 26 × 103; 2, 52 × 103; 3, 56 × 103; and 4, 78 × 103. The RNA which was not infectious consisted of four segments of mol. wt.: 1, 3.6 × 106; 2, 2.8 × 106; 3, 2.0 × 106; and 4, 1.3 × 106. The ribonucleoprotein (RNP) of TSWV, prepared by treatment of purified virus with Nonidet P-40, could be separated into three components by sedimentation on sucrose density-gradients. All three components contained protein 1; the slowest sedimenting component also contained RNA species 1, 2 and 4, the second component contained RNA species 1 and 2 and the fastest sedimenting component contained all four RNA species. The three components were slightly infectious and infectivity was enhanced by pooling these components. The RNP, when observed under the electron microscope, appeared to consist of strands.
Enveloped particles devoid of the outer layer of projections were obtained by treating TSWV with bromelain. These subviral particles retained about 1% of their original infectivity, had a density of 1.137 g/ml in sucrose and only contained protein 1. It was concluded that this protein forms the subunit of the nucleocapsid and that TSWV, unlike the majority of enveloped viruses, does not possess an internal membrane protein.
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Localization of Genetic Information Involved in the Replication of Alfalfa Mosaic Virus
More LessSUMMARYCowpea protoplasts were inoculated with different combinations of the four nucleoprotein components of alfalfa mosaic virus (AIMV), and the synthesis of viral particles, coat protein and RNA was analysed. Synthesis of viral particles and coat protein was detectable by polyacrylamide gel electrophoresis only when the inoculum contained the components B, M and Tb, accommodating RNAs 1, 2 and 3 respectively.
Upon inoculation with B + M + Tb, the synthesis of viral RNA was detectable by annealing of radiolabelled RNA from protoplasts to virus-specific double-stranded RNA. On inoculation with B + Tb or M + Tb no significant amount of viral RNA was produced. After inoculation with B + M, however, the production of virus-specific RNA was about 40% of that induced by complete infection, indicating that genetic information encoded by RNAs 1 and 2 is involved in viral RNA replication. As a mixture of the genomic RNAs did not induce the synthesis of virus-specific RNA, it is concluded that coat protein is required for the RNA synthesis induced by the mixture of B and M nucleoproteins.
The observation that no coat protein is produced upon inoculation with B + M + Ta components indicates that the replicase activity induced by B + M does not replicate RNA 4, the messenger for coat protein present in Ta particles.
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Dimethyl Sulphoxide (DMSO) Disassembles Tobacco Mosaic Virus Predominantly from the 5′-end of the Viral RNA
More LessSUMMARYThe effect of dimethyl sulphoxide (DMSO) on the stability of native and EDTA-treated tobacco mosaic virus (TMV, Vulgare strain) has been reinvestigated using a variety of chemical and biochemical techniques. Contrary to earlier reports, we conclude that TMV rods behave as a heterodisperse population, exhibiting one of two modes of uncoating. More than 50% of the rods disassemble rapidly and extensively in a unique polar fashion beginning at the 5′-end of the viral RNA. The remainder of the rod population uncoats more slowly, less extensively, and exhibits substantial bidirectional exposure of the viral RNA, commencing at the 3′-terminus but proceeding for no more than 500 nucleotides before the major uncoating event again shifts to the 5′-ends of the particles. A portion of the coat protein gene and the region around the assembly initiation site on the viral RNA appears most resistant to uni- or bidirectional stripping; this is in contrast to previous reports. This complex, biphasic behaviour of the TMV rod population, which produces two broad and relatively ill-defined peaks of metastable nucleoprotein intermediates, may account for many of the inconsistencies prevalent in earlier work.
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Volumes and issues
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Volume 105 (2024)
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Volume 103 (2022)
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Volume 102 (2021)
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