- Volume 49, Issue 2, 1980
Volume 49, Issue 2, 1980
- Bacterial
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Typing Mycobacterium tuberculosis with Mycobacteriophage Bo4
More LessSUMMARYMycobacteriophage B04 grown on the indicator host strain Mycobacterium fortuitum SN203 was restricted and modified by Mycobacterium tuberculosis H37Rv. Phage B04.H37Rv was restricted and modified by the alternate host SN 203. M. tuberculosis strain Myc 1025 was described as a r−m− isolate. By using the mycobacterial prototype strains for phage typing wild M. tuberculosis isolates, it was demonstrated that only the modified phage B04.H37Rv was a potential typing phage.
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Spectrophotometric Characteristics of Cholera Phage ϕ2 DNA
More LessSUMMARYPurified preparations of cholera bacteriophage ϕ2 were treated with cold phenol and the nucleic acid examined for its hydroxyapatite chromatographic pattern, thermal denaturation profile, base composition and mol. wt.
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- Animal
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Mapping of the Thymidine Kinase Genes of Type 1 and Type 2 Herpes Simplex Viruses using Intertypic Recombinants
More LessSUMMARYThe thymidine kinase induced in lytic infection by each of 36 intertypic recombinants of herpes simplex virus (HSV) type 1 and type 2 was identified as type 1 or type 2 by studies on the thermolability of enzyme activity, neutralization with type 1 or type 2 antiserum and agar gel immunodiffusion with type 1 or type 2 thymidine kinase antiserum. Fourteen recombinants induced no thymidine kinase, 12 induced type 1 thymidine kinase and 10 induced type 2 thymidine kinase. Correlation of these results with restriction endonuclease analysis of the DNA of the recombinants with five restriction endonucleases (XbaI, EcoRI, HpaI, HsuI, and BglII) allowed mapping of the type 1 thymidine kinase gene at 0.300 to 0.309 map unit and the type 2 thymidine kinase gene at 0.295 to 0.315 map unit.
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Isolation and Properties of Viruses from Poultry in Hong Kong which Represent a New (Sixth) Distinct Group of Avian Paramyxoviruses
More LessSUMMARYEight viruses isolated in Hong Kong were shown to be serologically related. One was obtained from the tracheal swab of a chicken and four were from cloacal swabs of ducks sampled at a poultry dressing plant. Three isolations were made from samples taken at a duck farm: two from pond water and one from faeces. Representatives of these isolates were shown to be paramyxoviruses but were serologically distinct from other avian and mammalian paramyxoviruses by haemagglutination inhibition and neuraminidase inhibition tests. Slight variations were seen in the properties of three isolates examined in detail. All three were apathogenic for chickens. The structural polypeptides of one isolate, PMV-6/duck/Hong Kong/199/77, were examined by SDS-polyacrylamide gel electrophoresis. Seven polypeptides were detected, with mol. wt. 180000, 76000, 60000, 55000, 51000, 48000 and 40000. The isolates represent a sixth serologically distinct avian paramyxovirus group.
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DNA Binding and Unwinding Activities Associated with Intracytoplasmic A Particles Isolated from Mouse Mammary Tumours
More LessSUMMARYIntracytoplasmic A particles (CAP), previously identified as probable cytoplasmic nucleocapsid precursor structures to mouse mammary tumour virus (MMTV), possess both DNA binding and DNA unwinding activities. CAP proteins bind to both single-stranded (ss)- and double-stranded (ds)DNA, with the ssDNA slightly preferred. This activity was linear over a 30-fold concentration of A particle protein and was not affected by NaCl concentrations as high as 0.6 m or pH changes over a wide range. DNA binding by CAP proteins was sensitive to heat or addition of sodium dodecyl sulphate (SDS) and was neutralized by pre-incubation of CAP with anti-MMTV p14, but not by anti-MMTV p27, p10 or anti-mouse casein. Incubation of CAP with dsDNA led to unwinding of the double helix as measured by its increased sensitivity to S1 nuclease digestion. This activity was also linear over a several-fold concentration of A particle protein and was heat labile. It was not inhibited by pre-incubation of CAP with either anti-MMTV p14 or with anti-MMTV, anti-MMTV p27 and anti-MMTV p10. DNA unwinding was inhibited by anti-A particle antiserum and to a lesser extent by anti-CAP p20-18.
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Characterization of the Non-permissive Infection of Rabbit Cornea Cells by Vesicular Stomatitis Virus
More LessSUMMARYRabbit cornea cells (RC-60) restrict the reproduction of vesicular stomatitis virus (VSV) (Thacore & Youngner, 1975). In cells infected with VSV alone, an inhibition in the synthesis of VSV genome RNA is observed. A number of parameters which could affect virus RNA synthesis have been examined: virus transcription and post-transcriptional modification, translation, modification of proteins and migration of the G protein to the surface of the cell; they all appear to be normal, although somewhat diminished, in the restricted system. In these cells, therefore, it is the replication of VSV RNA that is directly inhibited, although limited synthesis of both (+) and (-) strand genome length RNA does occur. When the cells are co-infected with rabbit poxvirus (RPV) as a helper virus, however, VSV production is normal. Our studies suggest that RPV plays a role in the maturation as well as in the replication of VSV RNA in RC-60 cells. Certain mutants of RPV have been found to lack the helper function and are unable to convert the RC-60 cells into a permissive host for VSV. These mutants should facilitate the elucidation of the mechanism by which RPV is able to overcome the restriction in these cells.
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Growth and Release of Several Alphaviruses in Chick and BHK Cells
More LessSUMMARYThe growth and release of several alphaviruses, including several strains of Sindbis virus (the wild-type strain, the large plaque and small plaque variants of the HR strain, and the HR mutant ts103), Semliki Forest virus (SFV) and Middelburg virus, and of the unrelated rhabdovirus, vesicular stomatitis virus (VSV), have been compared in chick cells and in BHK-21 cells as a function of the culture conditions for the host cell and the ionic strength of the medium. The small plaque strain of Sindbis HR, as well as SFV, grew better in BHK cells, whereas the large plaque strain of Sindbis HR showed a preference for chick cells. Wild-type Sindbis and VSV grew equally well in either cell. The optimum ionic strength for virus production as well as inhibition of virus release into the medium at low ionic strength depended upon both the virus and the host cell. Thus, VSV grown in medium of low ionic strength gave no additional release of virus on incubation with hypertonic medium (minimum effect), whereas ts103 released very little virus without exposure to hypertonic conditions (maximum effect). The viruses could be ordered as follows: minimum effect = vesicular stomatitis virus < Middelburg virus < Semliki Forest virus < Sindbis wt < Sindbis HR (large plaque) < Sindbis HR (small plaque) < Sindbis ts103 = maximum effect. After several passages in culture, chick cells required hypertonic conditions for optimum production and release of Sindbis virus. Furthermore, BHK cells cultured in different media responded differently to ionic strength for virus production and release. These results suggest that there is a charge-dependent step in the maturation of alpha-viruses, possibly a configurational rearrangement of glycoprotein E2 upon its formation from the precursor PE2, which is sensitive to the ionic strength of the medium, to the composition of the host plasmalemma and to differences in the virus glycoproteins.
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Cell-mediated Immunity in Herpes Simplex Virus-infected Mice: Functional Analysis of Lymph Node Cells During Periods of Acute and Latent Infection, with Reference to Cytotoxic and Memory Cells
More LessSUMMARYThe functional characteristics of lymphoid cells were investigated during acute and latent infection of mice with herpes simplex virus (HSV). Cytotoxic T cells were found in the draining lymph node (DLN) 4 days p.i. and had reached maximum activity between 6 and 9 days. After the 12th day and during the period of latent infection (> 20 days) no cytotoxic cell activity was observed. Cytotoxic activity could only be detected when the lymphoid cells had been cultured for a period of 3 days. In general, the cell killing was specific for syngeneic infected target cells, although some killing of uninfected targets was observed. In contrast to the cytotoxic response, DLN cells responding to HSV in a proliferation assay were detected towards the end of the acute phase and at least up to 9 months thereafter. The significance of these observations for the pathogenesis of HSV is discussed.
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The Characteristics of the Cell-free Translation of mRNA from Cells Infected with the Herpes Virus Pseudorabies Virus
More LessSUMMARYThe translation in vitro of mRNA from pseudorabies virus infected cells was studied using systems derived from wheat germ and from rabbit reticulocyte. The mRNA was shown by molecular hybridization to contain sequences complementary to virus DNA. Products of in vitro translation co-migrating with virus proteins on polyacrylamide gel electropherograms were detected and the major capsid protein (mol. wt. 150000) was shown to be present among the products by immune precipitation. Optimum conditions for the stimulation of amino acid incorporation in vitro were determined and found to be similar for mRNA from both infected and mock-infected cells.
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Ebola Virus: Identification of Virion Structural Proteins
More LessSUMMARYPolyacrylamide gel electrophoresis of purified Ebola virus revealed the presence of four major virion structural proteins which we have designated VP1, VP2, VP3 and VP4. Vesicular stomatitis virus (VSV) proteins were used as mol. wt. markers, and the virion proteins were found to have mol. wt. of 125000 (VP1), 104000 (VP2), 40000 (VP3) and 26000 (VP4). VP1 was labelled with glucosamine and is probably a glycoprotein. The density of the Ebola virion was approx. 1.14 g/ml in potassium tartrate. Virus nucleocapsids with a density of 1.32 g/ml in caesium chloride were released when virions were treated with detergents. Proteins VP2 and VP3 were consistently associated with released nucleocapsids and are probably the major structural nucleocapsid proteins analogous to the N protein of VSV. Protein VP4 was reduced or absent in released nucleocapsids and is probably analogous to the membrane (M) protein of VSV and similar viruses. The glycoprotein (VP1) is larger than the glycoprotein of any known negative-strand RNA virus and is not labelled well with 35S-methionine. VP1 is solubilized by detergent treatment, suggesting that it is a component of the virion spikes and analogous to the G protein of VSV. Our results, in conjunction with analysis of Ebola virion RNA (Regnery et al., 1980), strongly suggest that the virus is a negative-strand RNA virus and, along with Marburg virus, may constitute a new taxon within this group.
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The Replication of Type A Influenza Viruses in the Infant Rat: A Marker for Virus Attenuation
More LessSUMMARYTwenty recombinant influenza virus strains bearing HSw1N1, H1N1 or H3N2 surface antigens, together with their respective wild-type or laboratory-propagated parent viruses, were inoculated into 2 day-old infant rats and their replication in the turbinates and lungs of these animals observed over a period of 5 days. In addition, the ability of each of the recombinant and parent viruses to enhance a subsequent infection of these infant rats by Haemophilus influenzae type b was determined. The results showed that both parent and recombinant viruses replicated less well in the lungs than in the turbinates of infant rats, but the titres in both tissues were generally lower for the recombinant strains. The capacity of the majority of the recombinant influenza viruses to promote bacterial infection of the infant rats, as determined by the incidence of H. influenzae bacteraemia and meningitis, was also markedly less than that of their parent viruses. A correlation between virulence for man and both the replication in infant rat turbinates and the ability to enhance H. influenzae infection, was established for the virus strains studied. The data are discussed in relationship to the value of the infant rat-H. influenzae system as a laboratory marker for the determination of the virulence of influenza virus strains.
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A Fast Replica Plating Technique for the Isolation of Post-integration Mutants of the Moloney Strain of Murine Leukaemia Virus
More LessSUMMARYSeven temperature-sensitive (ts) mutants of Moloney murine leukaemia virus (Mo-MuLV) were isolated using a rapid, non-selective replica plating technique designed to select for post-integration mutants. Thymus-bone marrow (TB) cells, infected with mutagenized virus, were cloned and incubated at the non-permissive temperature (39 °C) for 10 days. The resulting colonies were screened for production of virus by replica plating supernatant from the ‘master’ tray on to a second tray pre-seeded with fu-1 (a cell line derived from L8 myoblasts) indicator cells. The ‘master’ tray was shifted to the permissive temperature (34 °C) for 48 h, then re-screened for virus production. Any colony on the ‘master’ tray which produced syncytia-inducing virus at 34 °C but not at 39 °C was potentially producing a ts mutant. Preliminary characterization by shift-down experiments and scanning electron microscopy of three of the ts mutants isolated by this technique revealed a mutant blocked before budding, one blocked at an early stage in the budding process and one with a defect after release of the virus.
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A Non-virion Surface Antigen on Moloney Sarcoma Virus-transformed Non-producer and Producer Cells
More LessSUMMARYSera from STU mice bearing sarcomas induced by cells producing the Moloney sarcoma-helper-virus (M-MSV/MLV) complex were cytotoxic for these cells as well as for M-MSV non-producer and M-MLV producer cells. Analysis by polyacrylamide gel electrophoresis of 125I-labelled surface antigens immunoprecipitated with such sera revealed the virus envelope glycoprotein gp71 on the producer cells and an additional antigen of mol. wt 55 K on the M-MSV-transformed producer and non-producer cells. This antigen was not found on non-transformed M-MLV-producing cells and was neither related serologically to structural polypeptides of murine C-type viruses nor to components of embryonal STU mouse fibroblasts and foetal bovine serum.
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The Detection of Intracellular Retrovirus-like Entities in Drosophila melanogaster Cell Cultures
More LessSUMMARYA Drosophila melanogaster cell line has been examined for the presence of retrovirus particles. When these cells were disrupted and analysed on sucrose density gradients a subcellular fraction with a density of 1.22 g/ml was found to possess endogenous DNA polymerase activity and could catalyse polymerization of deoxynucleotide triphosphates in response to added template primers. The latter activity had the cation and template primer responses expected for reverse transcriptase. A high mol. wt. polyadenylic acid-containing RNA was also purified from this fraction and could be dissociated by heat treatment into 30 to 35S and smaller species. Electron microscopy revealed the presence of torroidal forms reminiscent of intracytoplasmic A-type retrovirus particles within the Drosophila cells. Similar forms were found associated with the subcellular fraction of 1.22 g/ml. We conclude that our D. melanogaster cell line contains retroviruses similar, but not identical, to the A-type particles previously described in mammalian and avian cells.
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The Effect of Polyamines on Herpes Simplex Virus Type 1 DNA Polymerase Purified from Infected Baby Hamster Kidney Cells (BHK-21/C13)
More LessSUMMARYThe DNA polymerase whose synthesis is directed by the herpes simplex virus type 1 (HSV-1) DNA was purified 545-fold from BHK-21/C13 cells 16 h after infection with the virus. Spermidine and spermine stimulated the activity of the polymerase over the concentration range 0.5 mm to 2.5 mm. This effect was enhanced with increased concentrations of polyamine, maximum stimulation being threefold and fourfold for spermidine and spermine respectively. The diamine, putrescine, had little effect on the enzyme at the concentrations used (0.25 to 1.25 mm).
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Effect of Immune Serum on the Establishment of Herpes Simplex Virus Infection in Trigeminal Ganglia of Hairless Mice
More LessSUMMARYAdministration of immune serum to herpes simplex virus (HSV)-infected hairless mice could not prevent acute infection in the trigeminal ganglia and the eventual establishment of latency. However, immune serum reduced the amount of free virus in the ganglion during the acute phase of the infection. It appears also that the amount of virus that can be reactivated in the latently infected ganglion is decreased. This was indicated by a prolonged reactivation time and by a reduced virus content of ganglion homogenates prepared after various periods of co-cultivation.
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Characterization of X-tropic Endogenous Retrovirus-specific RNA in Uninfected Tissues and Lymphosarcoma of BALB/c Mice
More LessSUMMARYAnalysis of endogenous X-tropic BALB/c retrovirus-specific RNAs in a BALB/c mouse tissue containing mostly non-dividing cells (liver), in two normal tissues having significant proportions of dividing cells (20-h regenerating liver and 12-day embryo) and in cells of a lymphosarcoma of BALB/c mice were carried out by determining the extent to which the RNAs from these tissues hybridized to the virus 3H-cDNA probe and by their relative sedimentation values in a sucrose gradient. RNAs from 20-h regenerating liver, 12-day embryo and lymphosarcoma, each containing a significant proportion of proliferating cells, showed 8 to 21% higher hybridization values than normal liver RNA. Differences in the exact size classes of virus-specific RNAs and, in their relative proportions were found to exist in the four different tissue types examined and no correlation between a specific RNA size-profile and the proliferating activity of a tissue could be detected.
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Structural Heterogeneity in p30 Molecules of Type C Viruses
More LessSUMMARYTryptic digests of p30 proteins from mouse type C viruses were subjected to cation-exchange chromatography. Structural heterogeneity of p30 molecules was seen in two specific areas of the peptide elution profiles. These hypervariable regions of p30 proteins were used to discriminate representative ecotropic (N- and B-tropic), xenotropic (alpha and beta) and amphotropic viruses.
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Differentiation of Rubella Virus Strains by Neutralization Kinetics
More LessSUMMARYThe neutralization of rubella virus was investigated and shown to proceed by first order kinetics over the first 10 to 15 min. A comparison of the rate constant of neutralization (K) for six strains of rubella virus was carried out over this period of the reaction. Two particularly antibody-sensitive isolates were detected. It was noted that the three strains isolated from in utero infections were poorly neutralized by heterologous antisera when compared to postnatal strains.
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The Effect of Δ-9-Tetrahydrocannabinol on Herpes Simplex Virus Replication
More LessSUMMARYBoth herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) failed, in an identical fashion to replicate and produce extensive c.p.e. in human cell monolayer cultures which were exposed (8 h before infection, at infection, or 8 h p.i.) to various concentrations of Δ-9-tetrahydrocannabinol. Similar results were obtained with a plaque assay utilizing confluent monkey cells. Possible mechanisms for this antiviral activity are discussed.
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A Comparative Study on the u.v. Resistance of Double-stranded and Single-stranded Encephalomyocarditis Virus RNAs: Evaluation of the Possible Contribution of Host-mediated Repair
More LessSUMMARYTo reveal previously suggested host-mediated repair of u.v.-induced lesions in dsRNA of encephalomyocarditis (EMC) virus, two sets of experiments have been carried out: (i) samples of dsRNA of EMC virus were irradiated with different doses of u.v. light and their infectivity was assayed in Krebs II cells, before and after conversion of dsRNA into a ss form; (ii) samples of ssRNA of EMC virus were similarly irradiated and their infectivity was assayed before and after conversion of ssRNA into a ds form. No evidence for a significant host-mediated repair of dsRNA in this virus-cell system has been obtained.
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Simian Virus 40 and Moloney-Murine Sarcoma Virus Infection of Bona fide Mouse Epithelium
More LessSUMMARYMoloney-murine sarcoma virus (Moloney-MSV) and simian virus 40 (SV40) were found to infect successfully pure cultures of epithelial cells established from mouse liver and mammary tissue. MSV infection resulted in transient morphological foci with persistent production of infectious virus. SV40 infection produced detectable levels of virus-specific T antigen in the cells but morphological transformants were not observed.
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An Interferon-induced Refractory State of L Cells: Role of the Nucleus and Cytoplasm
More LessSUMMARYPre-treatment of L cells with 750 units/ml of mouse interferon depressed the yield of interferon when the cells were treated with Newcastle disease virus (NDV). The refractory state persisted for more than 5 days. Nucleated fragments (karyoplasts) obtained by cytochalasin B-induced enucleation were made from such interferon pre-treated cells; cells reconstituted from these karyoplasts produced less interferon in response to NDV than did cells reconstituted from karyoplasts derived from untreated cells. The source of the enucleated cytoplasm (cytoplasts) used in the cellular reconstitutions did not affect the yields of interferon.
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Derivation of a New Strain of Cowpea Chlorotic Mottle Virus from Resistant Cowpeas
More LessSUMMARYA new strain (R) of cowpea chlorotic mottle virus (CCMV) was isolated from plants of the resistant cowpea plant introduction 186465 which were inoculated with the type strain (T). Strain R was serologically different from five naturally occurring CCMV strains. Furthermore, it was able to replicate in and systemically invade cowpea plants which were resistant to strain T. Pseudorecombinant studies with the RNAs of strains T and R established that RNA 1 controlled systemic invasion of the resistant cowpea cultivar. Both RNA 1 and RNA 3 influenced replication in resistant cowpeas. RNA 3 also controlled the composition of the coat protein, while RNA 1 influenced symptoms in susceptible cowpeas.
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Cytopathic Changes in Leaf Tissue of Gynura aurantiaca Infected with the Viroid of Citrus Exocortis Disease
More LessSUMMARYTissue from symptom-bearing young leaves of Gynura aurantiaca plants systemically infected with citrus exocortis viroid (CEV) was investigated with the electron microscope. It was found that, in contrast to a previous report, the appearance of paramural bodies or plasmalemmasomes cannot be considered to be the primary c.p.e. in G. aurantiaca because these structures are also present at the same frequency in healthy control plants. The plasmalemmasomes in healthy tissue, characterized by vesicular or tubular internal structures, are found at different developmental stages and in the process of spreading between the plasmalemma and cell wall of developing young cells. The viroid-specific cytopathic changes are alterations of the plasmalemmasomes themselves, which may make it difficult to relate them to the corresponding normal structures. The malformed plasmalemmasomes were consistently found in association with the extremely distorted cell walls characteristic of viroid-infected tissue; spreading of plasmalemmasomes was not observed. These findings indicate an intimate functional relationship between plasmalemmasomes and cell wall formation which is disturbed as a consequence of viroid infection.
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Further Observations on the Structure of Particles of Potato Virus X
More LessSUMMARYX-ray diffraction studies of oriented specimens of potato virus X, and optical diffraction from electron micrographs of individual particles, are consistent with a helical structure which repeats in eight turns of the helix as proposed by Horne et al., (1975). The studies further suggest that there are 8q + 7 protein subunits in the repeat period, and that the probable number of subunits in eight turns is 71.
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