- Volume 16, Issue 2, 1972
Volume 16, Issue 2, 1972
- Articles
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The Selective Release of Proteins from a Bovine Enterovirus
More LessSUMMARYA bovine enterovirus (serotype VG-5-27) was grown in BHK cells and purified by sedimentation procedures. Virus particles were heated at 100° in the presence of urea-sodium dodecyl sulphate, urea or borate buffer at pH 10. Whereas complete degradation of the virus in the presence of urea-SDS showed four main polypeptides, namely VP1, VP2, VP3 and VP4, heating in the presence of urea or alkali alone resulted in the selective release of VP2 and VP4, predominantly. Electron-microscopic examination of the residual complexes showed evidence for unstable intermediate ring-like particles and also fibrous material. These complexes lacked RNA and contained mainly VP1 and VP3. We interpret the results as indicating that the polypeptide VP2 may have a specific location at the apical region of the capsid.
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Haemagglutinin Relationships of Hong Kong(H3) and Asian(H2) Influenza Strains Delineated by Antigen-Specific Recombinants
More LessSUMMARYReports by others have described the haemagglutinins of Hong Kong (H3N2) strains and the Asian (H2N2) strains as being antigenically unrelated. Cross reactions seen by the haemagglutination-inhibition (HI) test have been attributed to antibodies to a second major surface antigen, the neuraminidase, which is common to both subtypes. However, observations on human anamnestic antibody responses suggest the two haemagglutinins are related. In the present study we reexamined the antigenic relationship of these viruses using haemagglutinin specific virus recombinants, chicken and ferret antisera, antibody equilibrium filtration methods, and cross-infection in ferrets. Data from these studies consistently demonstrated an asymmetric relationship, independent of the neuraminidase, between the H2 and H3 strains.
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The Kinetics of Haemagglutination by Semliki Forest Virus: A New Sedimentation-Enumeration Method
More LessSUMMARYA novel method is described for the quantitative study of haemagglutination in terms of the continuous and direct sizing and counting of aggregates of red blood cells (RBC) as they settle in free suspension. This sedimentation-enumeration (SE) method was used to estimate the concentration of haemagglutinating particles in terms of the number of new RBC-RBC bonds formed. In haemagglutination by Semliki Forest virus (SFV), the formation of RBC-RBC bonds is interpreted in terms of two competitive and near first-order reactions: the rate of inactivation of SFV haemagglutinin at low values of pH, and the rate of adsorption of residual SFV haemagglutinin by RBC. The SE method provides an estimation of the concentration of virus in terms of haemagglutinating activity which is independent of container wall effects and of the concentration of RBC. Results were compared with parallel estimates of the concentrations of infective and physical particles. At the optimum pH 6.3 for haemagglutination, and at a concentration of 107 RBC/ml., about 7 particles of haemagglutinin were required for the formation of one RBC-RBC bond.
At low concentrations of SFV haemagglutinin, the distribution of single RBC and of aggregates of RBC was consistent with a statistical-mechanical theory of aggregation which provides a basis for the interpretation of the mechanism of haemagglutination. The distributions observed in this study were not consistent with the ‘dimers-only’ hypothesis of Levine, Puck & Sagik (1953) which was used by Cheng (1961) in an early study of haemagglutination by SFV.
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Electron Microscopy of Maize Rough Dwarf Virus Assembly Sites in Maize. Cytochemical and Autoradiographic Observations
More LessSUMMARYThe following experiments have been carried out: (1) enzymatic digestion with pronase and RNase, to study the nature of the cytoplasmic inclusions (viroplasm and cytoplasmic tubules) induced by the virus, and of the smaller virus particles as seen in the viroplasm; (2) autoradiographic experiments after [H3]-uridine incorporation, to detect the possible sites of virus synthesis and/or assembly.
Enzymatic digestion of maize leaf tumours caused by maize rough dwarf virus (MRDV) showed that the viroplasm and cytoplasmic tubules induced by the virus are composed mainly of protein, and that the small particles embedded in the viroplasm are ‘naked’ RNA particles, since they were promptly digested by RNase, while the complete particles were not.
Autoradiographic experiments demonstrated that [H3]-uridine was incorporated almost exclusively into the viroplasm containing virus RNA particles, while no incorporation was detected in the viroplasmic regions free of virus particles and in the mature virus particles as seen in the cytoplasm.
It is concluded that the viroplasm is most probably the site of assembly of MRDV.
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The Structure of Single-Stranded Virus RNA in situ. A Study of Absorption Spectra and Optical Rotatory Dispersion of Tobacco Mosaic Virus and Potato Virus X Preparations
More LessSUMMARYThe secondary structure of RNA in particles of two plant viruses with helical symmetry (tobacco mosaic virus (TMV) and potato virus X (PVX)) was studied by determining their RNA extinction spectra in situ. The true values of extinction of light-scattering virus suspensions were calculated by extrapolation. To determine the degree of hyperchromicity of internal RNA, the spectra of suspensions of intact and disrupted TMV and PVX were compared. Virus particles were disrupted by heating or by sodium dodecyl sulphate. The contribution of protein to the total extinction of virus was taken into account during calculations of hyperchromicity of internal RNA. It was shown that RNA in particles of both viruses is in a fully hyperchromic state. The interaction of both internal RNA's with formaldehyde was also investigated. The reaction of RNA in situ with formal-dehyde was followed by the increase in extinction of the virus suspensions in the 280 to 290 nm. spectral region. It was found the amino-groups of the bases of internal PVX RNA readily react with formaldehyde. On the other hand, the TMV RNA in situ was completely resistant to formaldehyde. A substantial difference in the optical rotatory dispersion (ORD) curves of two viruses was also revealed. The differences in reactivity with formaldehyde and in ORD curves of TMV and PVX probably reflect the different character of RNA-protein interactions in particles of the two viruses.
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Heterogeneity in the Phospholipid Content of Purified Rabies Virus (era Strain) Particles
More LessSUMMARYWhen rabies virus (era strain) was purified by precipitation with zinc acetate, Sephadex filtration, and centrifuging in a sucrose density gradient, usually about half of the virus particles spontaneously lost a portion of their envelope phospholipids. Because of differences in buoyant density, partially delipidized virus particles (about 1.16 g./cm.3) can be separated from intact virus (about 1.14 g./cm.3) by centrifuging in a sucrose density gradient. High egg passage virus (flury strain) purified by the same procedure, did not exhibit this type of heterogeneity. The release of phospholipids from era strain virus particles did not cause a marked decrease in the infectivity of the virus, but the morphology of the virus envelope changed from bullet- to bag-shaped. The protein (glycoprotein) composition and the RNA contents of intact and partially delipidized virus forms were similar.
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Biochemical Changes during Mixed Infections with Bacteriophages T2 and T4
More LessSUMMARYBiochemical changes accompanying the partial genetic exclusion of wild-type T2 genes in mixed infections with various mutants of phage T4 have been examined. Total phage yields varied between 14 and 60% of those observed after infections by either wild-type phage alone. DNA synthesis was delayed, but then occurred at rates close to those found with each wild-type phage strain. Among the progeny virus, the frequencies of the excluded wild-type T2 genetic markers ranged between 0.02 and 0.45, and varied with the markers' positions on the genetic map. Measurement of the frequencies of markers among the progeny phage at 15 to 60 min. after infection showed that the weakly excluded T2 genes 1 and 20 increased slightly, while the strongly excluded T2 genes 33 and 42 dropped sharply and then remained constant. The strongly excluded T2 genes 42 and 56 formed low levels of their respective products deoxy-CMP-hydroxymethylase and deoxy-CTP-ase in mixed infections with the corresponding T4 amber mutants in a non-suppressor host. The T2-induced enzymes thymidylate synthetase and lysozyme reached normal levels during infections with the corresponding defective T4 strains. With mixed infections in an endonuclease-I deficient host, [32P]-labelled T2 phages yielded 14% of their DNA as acid-soluble products, while no breakdown of [32P]-labelled T4 DNA was detected. The possibility is discussed that partial exclusion results from T4-induced nuclease action against T2 chromosomes followed by marker rescue of T2 DNA fragments with intact T4 genomes.
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Relationship Among Temperate Agrobacterium Phage Genomes and Coat Proteins
More LessSUMMARYFive temperate Agrobacterium phages, two of which are defective, and one presumed temperate, phage PS8, isolated from crown gall tissue, were prepared, purified and compared.
The four phages, omega, PS8, PB2A and LV-1, were indistinguishable. They possess a hexagonal head of about 70 nm. with a flexible tail about 200 nm. long. Their DNA had a T m of 92.7° ± 0.1°, a % GC of 56.7 ± 0.2, a mol. wt of 34.1 × 106 and comprised the four normal bases A, T, G and C. No evidence was found for repetitive units in PB2A. These four genome DNA types gave 100% hybridization (limit of error 6%). The electrophoretic protein profiles on SDS polyacrylamide gels are essentially identical: four major bands with similar mol. wt. About half of the phage protein has a mol. wt of 48,000 (perhaps a major head protein), about one-third has a mol. wt of 30,000, one sixth has a mol. wt of 15,500 and 4% has a mol. wt of 69,000. No omega-type prophage DNA was detectable in the cured bacteria v-ic by DNA:DNA hybridization. The latter bacteria were as pathogenic as the prophage-containing parent strain v-i, which throws some doubt on the role of the omega phage group in crown gall induction.
The phage-like particle P0362, isolated from the pathogenic Agrobacterium tumefaciens 0362 had a 60 to 70 nm. head and a straight 130 nm. tail. Its DNA was double-stranded with a slightly lower T m of 92.4°, a mol. wt of 25 × 106. The electrophoretic profiles of coat proteins differed from those of the other phages. Its DNA is less than 17% homologous with the omega group. The eventual role of this phage in crown gall induction remains to be established.
The phage-like particle P8149, isolated from the non-pathogenic Agrobacterium radiobacter ncib 8149, was quite different. The bipyramidal head was much smaller (40 nm. diameter) and had a very short bud-like tail. The DNA was double-stranded, with a T m of 93.9°, a mol. wt of 10 × 106. The electrophoretic profile of the coat proteins differed from those of the other phages. The DNA does not hybridize with the omega group. As this phage was isolated from a non-pathogenic A. radiobacter, it probably has no function in crown gall. A. radiobacter 8149 is bilysogenic.
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Ultrastructure of Mycoplasmatales Virus Laidlawii 1
More LessSUMMARYThe morphology and ultrastructure of Mycoplasmatales virus laidlawii 1, a new helical DNA virus, were studied by electron microscopy and optical diffractometry. Numerous small unenveloped rod-like particles and a few longer forms were seen in purified, concentrated preparations of infective virus, and associated with virus-infected mycoplasmal cells in culture. Observations on these virus particles are discussed in relation to their structure.
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Defective Mouse Sarcoma Virus Deficient in DNA Polymerase Activity
J. T. May, K. D. Somers and S. KitWe reported previously the clonal isolation in normal rat kidney (NRK) cells of two variants of mouse sarcoma virus (MSV) from the Moloney sarcoma-leukaemia complex (MSV-MoLV) (Somers & Kit, 1971). One isolate (MSV-1) has a restricted host range, is capable of transforming NRK cells, but cannot replicate infectious progeny virus. The other isolate (MSV-6) is incapable of either transformation or replication in any cell tested. Recently, it was reported that non-infectious Rous sarcoma virus [RSVα(0)] was deficient in RNA-dependent DNA polymerase activity, but conflicting evidence was reported for DNA-dependent DNA polymerase (Hanafusa & Hanafusa, 1971; Robinson & Robinson, 1971). We examined both DNA polymerase and endonuclease activities in purified particles of MSV-MoLV, MoLV, and in the two clonal isolates, MSV-1 and MSV-6. All preparations contained similar levels of endonuclease activity. However, the DNA polymerase activities of MSV-1 and MSV-6 were 25% and 3%, respectively, of the activity found in MSV-MoLV or MoLV.
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Inability of Isoquinoline Derivatives to Inhibit Virus Neuraminidase Activity
More LessThe antivirus activity of isoquinoline derivatives, 1-(p-chlorophenoxymethyl)-3, 4-dihydroisoquinoline hydrochloride (compound I) and 1-(p-methoxyphenoxymethyl)-3, 4-dihydroisoquinoline hydrochloride (compound II) has been demonstrated by many studies in vitro and in vivo on several agents of human respiratory diseases. The mechanism by which these compounds inhibit the multiplication of influenza viruses was presumed to be due to their antineuraminidase activity (Tute et al. 1970).
During an investigation on the antivirus effect of isoquinoline derivatives, we found that both derivatives did not inhibit the enzyme reaction but blocked only the colour development of N-acetylneuraminic acid (NANA). This brief report will be concerned with the results obtained regarding this problem.
Influenza virus A2/japan-305 (Chiba), partially purified from infected chorioallantoic fluid, was used as a virus enzyme source. The enzyme activity was measured by a slight modification of the method described by Rafelson, Gold, & Priede (1966).
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A Quantitative, Single-Radial-Diffusion Test for Immunological Studies with Influenza Virus
More LessAntibodies for influenza haemagglutinin and neuraminidase are assayed routinely in haemagglutination-inhibition (Hirst, 1942) and neuraminidase-inhibition (Ada, Lind & Laver, 1963) tests which involve reaction systems of three components. More recently, influenza antibodies have been detected by gel immuno-double-diffusion tests with influenza viruses disrupted by detergents (Styk & Hanna, 1966; Schild & Pereira, 1969). Although the latter method has the advantage that it detects antibodies for each of the major antigens of the virus in a single test system (Schild et al. 1972a; Schild, 1972) the quantitation of antibody is not readily achieved. In the present report we describe a quantitative, single-radial-diffusion test which is a sensitive, rapid and convenient method for the estimation of antibodies to the haemagglutinin and neuraminidase antigens of the influenza virus. A unique feature of this test is that only one component, the antibody, diffuses during the test; the other component, the antigen in the form of intact influenza virus particles, does not diffuse.
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Recombination in Staphylococcal Bacteriophages
More LessThe only published works dealing with recombination amongst staphylococcal bacteriophages are those of Rountree & Asheshov (1961) and Wentworth & Romig (1968). These authors have demonstrated the existence of a defective lysogen by studying the recombination between invading bacteriophages and the defective lysogen. However, until now, the lack of suitable genetic markers has hampered more detailed study of staphylococcal bacteriophage recombinants. This report deals with recombination between prophages and invading bacteriophages having recognizable genetic traits associated with (a) their ultrastructure, (b) their specificity as seen by superinfection immunity, and (c) their ability to suppress the lipase reaction of host staphylococci as seen on egg yolk medium (i.e. their ability to convert an egg yolk positive (EYP) reaction to a negative (EYN) one upon lysogenization). Table 1 summarizes the characteristics of the three phages used in the present work.
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Haemolysis by Sendai Virus: Lack of Requirement for Neuraminidase
More LessSince both neuraminidase and the haemolytic activity of Sendai virus were inhibited by Cu2+ ions, it was suggested that neuraminidase was essential for haemolysis (Howe & Morgan, 1969). Haemolysis by Newcastle disease virus (NDV) appeared to be intimately associated with the capacity of the virus to become irreversibly attached to the cell (Burnet, 1950), indicating that neuraminidase, responsible for elution of the virus from red blood cells (RBC), was perhaps not required for, or may even have hindered haemolysis.
The results of this study, aimed at the elucidation of the role of neuraminidase in Sendai virus-induced haemolysis, show that: (1) treatment of the virus by disodium ethylenedi-aminetetraacetate (EDTA) or hydroxyethylethylenediaminetriacetate (HEDTA) causes a simultaneous decrease in neuramininidase activity and an increase in haemolytic activity of the virus. (2) 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DDANA), a reversible inhibitor of neuraminidase (Meindl & Tuppy, 1969; Meindl et al. 1971), does not interfere with, but augments haemolysis.
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Some Properties of Virus and Immune-Induced Human Lymphocyte Interferons
More LessSmall lymphocytes respond in vitro to virus infections with the synthesis of interferon. But viruses and their nucleic acids are not the only inducer. Recently we have demonstrated that specific antilymphocyte globulins (ALG) (Falcoff et al. 1972a; Falcoff, Oriol & Iscaki, 1972b), and its divalent F(ab′)2 fragments (Falcoff et al. 1972b), are also able to induce interferon synthesis. A number of unrelated non-virus substances share this property: phytohaemagglutinin (Wheelock, 1965), concanavalin (Falcoff, unpublished results), etc.
The most remarkable difference between the two groups of inducers is the nature of the target cell: while viruses are active on many kinds of cultured cells, including lymphocytes, the second group is only active on immunocompetent cells.
The present study was undertaken in an attempt to distinguish the antivirus substance synthesized by ALG-stimulated human lymphocytes from the interferon induced in similar cultures by irradiated Newcastle Disease Virus (NDV).
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