- Volume 52, Issue 2, 1981
Volume 52, Issue 2, 1981
- Animal
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Baculovirus Replication: Electron Microscopy of the Sequence of Infection of Trichoplusia ni Nuclear Polyhedrosis Virus in Spodoptera frugiperda Cells
More LessSUMMARYThe basic sequence of morphological development of Trichoplusia ni nuclear polyhedrosis virus (NPV) in Spodoptera frugiperda cells at a temperature optimal for virus replication has been determined. The development of this virus is similar to that reported for other baculoviruses with nuclear virogenic stroma preceding nucleocapsid, virus particle and polyhedron production; envelopment of nucleocapsids occurs de novo within the nucleus or by acquisition at the nuclear or plasma membranes. Cells in which virus DNA synthesis was arrested by blocking with cytosine arabinoside failed to show virus development beyond the appearance of a stroma without nucleocapsids. Cells in which virus-specified polypeptides were rendered non-functional by azetidine incorporation at prescribed periods delayed morphological change. This development is discussed in relation to the basic biochemical changes which occur in infected cells.
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Relationship between Herpesvirus ateles-associated Nuclear Antigen (HATNA) and the Number of Virus Genome Equivalents in HVA-carrying Lymphoid Lines
More LessSUMMARYA DNA-binding antigen (HATNA) was demonstrated in 7 out of 14 cell lines carrying Herpesvirus ateles (HVA) by the acid-fixed nuclear-binding technique. The seven HATNA-positive lines had means of 95, 96, 103, 177, 240, 343 and 326 virus genome equivalents/cell. For the seven HATNA-negative lines, the figures were 4, 8, 10, 33, 39, 72 and 110. This indicates a relationship between the number of HVA genome equivalents/cell and the detectability of HATNA. This association was independent of the virus producer status of the lines.
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Partial Purification and Characterization of Syrian Hamster Interferon
More LessSUMMARYSyrian golden hamster interferon was made by stimulating secondary or benzo(α)pyrene-transformed embryo cells with Newcastle disease virus. Titres of 1000 to 6000 units/ml of tissue culture fluid were obtained. The ionic properties of this interferon were characterized by chromatography on cation and anion exchangers and also by isoelectric focusing when two components were observed: a major component, pI 5.8 and a minor component, pI 6.6. Hamster interferon was partially purified on a tandem of sorbents of diverse chromatographic bias: anion exchanger → metal chelate → hydrophobic ligand. The purified preparation had a specific activity of about 1 × 106 units/mg protein; the recovery of activity was nearly complete. The apparent mol. wt. of hamster interferon, as estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions, is 17000. Crude Syrian hamster interferon preparations had some activity on mouse cells; this heterologous activity was entirely due to a small subpopulation of interferon molecules which could be isolated on phenylagarose.
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Coronavirus JHM: a Virion-associated Protein Kinase
More LessSUMMARYCoronavirus JHM contains six major proteins, one of which, the 60000 mol. wt. nucleocapsid protein pp60, is phosphorylated. In JHM-infected cells ip 60K, the intracellular precursor to pp60 is also phosphorylated. Associated with purified JHM virions is a protein kinase which will phosphorylate pp60 and a variety of exogenous substrates in vitro. The enzyme has the characteristics of a cyclic nucleotide-independent protein kinase. Both the in vivo reaction and the enzyme activity in vitro transferred the γ-phosphate of ATP to serine residues on the nucleocapsid protein.
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Defective Interfering Particles of Fixed Rabies Viruses: Lack of Correlation with Attenuation or Auto-interference in Mice
More LessSUMMARYSix different fixed strains of rabies virus were analysed for their capacity to produce defective particles following acute infection of BHK-21 cells. Five of the six strains produced one or more defective particle populations with strain-specific sedimentation properties, particle length and abbreviated RNA genome size. These defective particles varied in their capacity to interfere with replication of standard rabies virus in cell culture. Each virus strain characteristically either killed adult mice according to a normal dose-response pattern or to an auto-interference type of pattern, or failed to kill mice. Different strains also varied in their capacity to induce a cytopathic effect in cell culture. However, there was no apparent correlation between the presence of defective particles and the pathogenic potential of rabies virus in mice or in cell culture.
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A Quantitative Assay for Cytolysis Induced by Newcastle Disease Virus
More LessSUMMARYWe report here an assay for quantifying virus-induced lysis, in the absence of antibody and complement, produced within 2 h after adsorption. This technique makes use of 51CrO4 release from cell monolayers pre-incubated overnight with the isotope. The release of 51Cr is specific for virus-induced lysis and is suppressible by 0.001 m-Ca2+. This assay clearly distinguishes between wild-type Chinese hamster ovary (CHO) cells, clone K and a fusion-resistant mutant (CHO-15B), which was found to be resistant to virus-induced cytolysis. The stability of the association of isotope with monolayers of this cell type under the labelling conditions described makes this technique applicable to the study of the cytolytic effects of virus infection.
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In situ Electron Microscopical Observation of Cells Infected with Herpes Simplex Virus
More LessSUMMARYTransport and release of herpes simplex virus (HSV) in an African green monkey kidney cell line (CV-1) was followed by electron microscopy for up to 24 h p.i. Transmission electron microscopy and scanning electron microscopy were employed. For the former approach, electron microscopical autoradiography of whole cultured cells and in situ thin section techniques were used. The following new observations were made. (1) Except in peripheral parts of the cells, where the cytoplasmic membrane could make a ruffling movement relatively freely, virus particles were found only on the dorsal surface of the cell and not on the surface facing the substratum. By observation of thin sections in situ, it was confirmed that the virus particles within intracytoplasmic vacuoles were apparently released by a reverse phagocytic process from the cell surface adjacent to microvillus projections. (2) Progeny virus particles in the nucleus moved to the cell surface within 2 h after maturation.
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Antigenic and Structural Relatedness among Non-capsid and Capsid Polypeptides of Polioviruses belonging to Different Serotypes
More LessSUMMARYAntibodies were raised by immunization of Macaca fascicularis monkeys with extracts of M. fascicularis kidney cells separately infected with one of the three poliovirus serotypes. Preparations of antibodies were shown to be strictly type-specific in the neutralization test but formed immune complexes adsorbable on Staphylococcus aureus, Cowan I strain cells, with heterotypic as well as homotypic virus-specific polypeptides present in extracts from the virus-infected cells. The presence of intertypic antigenic determinants was demonstrated by this technique on both the non-capsid and capsid poliovirus polypeptides.
Structural variations of poliovirus polypeptides were studied by analysing products of their partial proteolysis. Non-capsid polypeptides encoded in the central portion of the virus genome (polypeptides 5b and X) as well as in the 3′-terminal region (NCVP2 and NCVP4) were found to be highly conserved, whereas capsid polypeptides VP1, VP2, and VP3, which are encoded in the 5′-terminal region of the virus RNA, displayed a much greater variability.
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Antiviral Properties of Polyinosinic Acids containing Thio and Methyl Substitutions
SUMMARYPolyinosinic acids containing methyl and sulphur substitutions are potent inhibitors of reverse transcriptase. Substitution of sulphur for oxygen at the 6 position produces significant effects on the properties of polyinosinic acid: the kinetics of inhibition change from competitive to mixed-type and the inhibition constant falls by three orders of magnitude. In contrast, 1-methyl substitution produces no such effects. Poly(1-methyl-6-thioinosinic acid) or poly(m1s6I) inhibits irreversibly, inhibiting all ten reverse transcriptases tested under a variety of assay conditions. In cell culture test systems, poly(m1s6I) is capable of blocking both infection by non-transforming viruses and transformation by a sarcoma virus. The presence of poly(m1s6I) in a preinfected culture results in the production of non-infectious virus particles lacking reverse transcriptase activity.
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Detection of Simian Virus 40 T-Antigen-related Antigens by a 125I-Protein A-binding Assay and by Immunofluorescence Microscopy on the Surface of SV40-transformed Monolayer Cells
More LessSUMMARYSimian virus 40 (SV40)-transformed cells express the SV40-specific tumour transplantation antigen (TSTA) on the cell surface and the SV40-coded tumour antigen in their nuclei. TSTA is defined by SV40-specific transplantation immunity, whereas T-antigen (T-Ag) can be detected serologically by indirect immunofluorescence. Both antigens, however, are derived from the A gene of SV40. We therefore analysed SV40-transformed cells for the presence of serologically detectable T-Ag-related molecules. Such antigens could not be detected on the surface of living SV40-transformed cells in monolayers. However, after a short formaldehyde fixation it was possible to stain the cell surfaces of SV40-transformed cells with sera from rabbits immunized with purified SDS-denatured T-Ag, but not with sera from hamsters bearing SV40-induced tumours. T-Ag-related antigens could be detected with both types of antisera by applying a more sensitive 125I-protein A assay. The T-Ag specificity of the binding of hamster SV40 tumour sera was demonstrated by a 125I-IgG-blocking assay in which preincubation of formaldehyde-fixed SV40-transformed cells with rabbit anti-SDS-T-Ag serum inhibited the binding of hamster SV40 tumour serum by about 70%. The localization of T-Ag-related antigens on the outside of plasma membranes of formaldehyde-fixed cells was shown by an anti-SDS-T-Ag serum-specific binding of fluorescein isothiocyanate-labelled Staphylococcus aureus to the cell surface. Our results are consistent with the hypothesis that SV40 T-Ag-related antigens are involved in the formation of TSTA.
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Baculovirus Replication: Stimulation of Thymidine Kinase and DNA Polymerase Activities in Spodoptera frugiperda Cells Infected with Trichoplusia ni Nuclear Polyhedrosis Virus
More LessSUMMARYTrichoplusia ni multiply enveloped nuclear polyhedrosis virus stimulates thymidine kinase and DNA polymerase activities in infected cells. The kinetics of induction and sensitivity of induction to cytosine arabinoside indicate that they represent early functions in infected cells. The basic characteristics of the induced enzymes are defined.
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Characteristics of a Persistent Rubella Infection in a Human Cell Line
More LessSUMMARYA persistent infection of the human MCF-7 cell line (MCF-7RV) was established with the DBS strain of rubella virus at a low multiplicity of infection. Fluorescent antibody staining revealed that 100% of the cells were positive for rubella antigens. The infected cells were refractory to superinfection with vesicular stomatitis virus (VSV) but were susceptible to herpes simplex virus type 2 (HSV-2). No interferon could be detected in infected cell culture fluid, and continuous passage in the presence of antibody did not lead to a decrease in the percentage of infected cells. Virus production in the persistently infected cells represented a 5- to 10-fold increase over primary infection. Plaque assays at 30 and 39 °C of the virus produced at 37 °C revealed the presence of temperature-sensitive (ts) mutants. If MCF-7RV cells were maintained at 30 °C, significant increases in virus production were observed, leading to cytopathic effect and destruction of the monolayer. If maintained at 39 °C, MCF-7RV cells produced less virus and demonstrated normal morphology. These data suggest that the naturally selected population of ts mutants being produced by these cells represents the mechanism by which persistence is maintained.
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The Interaction of Influenza Virus Haemagglutinin with Phospholipid Vesicles – Morphological and Immunological Studies
More LessSUMMARYHA—lipid spheres or ‘virosomes’ were prepared using neutral or negatively charged, but not positively charged, phospholipids. Virosomes were similar in size and shape to native virus particles although the HA subunits were at least twofold less numerous on the virosomes. The HA subunits were attached by their narrow end to the lipid bilayer, and could be removed by digestion with bromelain. However, HA subunits released from intact virus by digestion with bromelain, which removed the hydrophobic tail of the molecule, could not attach to liposomes. Measurements of HA spikes before (mean length 14.2 ± 0.9 nm) and after attachment to liposomes (mean length 13.3 ± 0.7 nm) and examination of freeze-fractured virosomes indicated that the HA did not penetrate deeply into the lipid bilayer. Similarly, HA subunits did not penetrate deeply into the lipid of virus particles. NP and M proteins could be attached to liposomes but could not be visualized by electron microscopy. Virosomes were taken up by Vero cells by viropexis with no evidence of fusion. Incorporation of HA or NP on to virosomes resulted in increased immunogenicity compared to free HA subunits or NP respectively. This adjuvant activity was not apparent in simple mixtures of HA and liposomes. The antibody induced by HA subunits, virions and virosomes reacted similarly with strain-specific (SS) antigenic determinants of the haemagglutinin.
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The Isolation of Large and Small Plaque Canine Distemper Viruses which Differ in their Neurovirulence for Hamsters
More LessSUMMARYLarge and small plaque-forming viruses were isolated from the Onderstepoort strain of canine distemper virus (CDV). Small plaque virus, which was released more slowly from infected cells than large plaque virus, readily established persistent infections in Vero cells, whereas large plaque virus required undilute passage to do so. All persistently infected cultures eventually released small plaque virus. No difference was found in the size of polypeptides induced by either plaque-purified viruses or virus released from persistent cultures. Both dilute and undilute passage, large plaque virus produced an acute neurological illness in weanling hamsters, whereas small plaque virus failed to produce any clinical signs of disease for 3 months after inoculation. After this period 50% of the animals infected with small plaque virus showed a general deterioration in their condition and lesions were observed in the brain which resembled those found in cases of large plaque virus infection. Serum-neutralizing antibody titres to CDV rapidly increased after infection with small plaque virus, whereas animals infected with large plaque virus had low or undetectable levels. All hamsters infected with small plaque virus and a small number which survived large plaque virus infection had elevated titres of antibody over a test period of 15 months.
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The Correlation of Fatty Acid Content of Infected Cells and Virions with Newcastle Disease Virus (NDV) Virulence
More LessSUMMARYChick embryo fibroblasts (CEF) infected with virulent strains of Newcastle disease virus (NDV) showed a dramatic increase in total unsaturated fatty acids (UFA). This increase was not seen in cells infected with the avirulent strains of NDV, Sendai virus or influenza A (PR8). The virions of the virulent strains of NDV harvested from the chorioallantoic cavity also had a higher UFA content compared to the avirulent ones. The kinetics of UFA increase in the virulent strains could be correlated with published data on the kinetics of RNA and protein synthesis inhibition, polykaryon formation and membrane permeability changes.
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Intermediate Size Papovavirus Particles in Pregnancy Urine
More LessSUMMARYBy electron microscopy of negatively stained urinary sediments, papovavirus particles of size intermediate between papillomavirus and polyomavirus have been detected in the urine of a pregnant woman. The excretion of size variants of these viruses and the absence of detectable papillomas in any of the women who were found to excrete only papillomavirus represent new findings.
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The Location of the Bromelain Cleavage Site in a Hong Kong Influenza Virus Haemagglutinin
More LessSUMMARYThe site of bromelain cleavage in the haemagglutinin of the Hong Kong influenza virus A/Memphis/102/72 has been determined by using a diagonal peptide mapping procedure on the thermolytic digest of amidated BHA. The data show that bromelain cleavage removes the C-terminal 46 residues from HA2, and that the new carboxyl-terminal residue of BHA2 is Gly 175. This is close to the beginning of the hydrophobic membrane-interacting sequence that starts at residue 183.
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Transcriptional Control of Endogenous Virus Genes in Murine Lymphocytes
More LessSUMMARYThe expression of endogenous retrovirus in murine lymphocytes is under genetic control and also depends on the differentiation state of the lymphocytes. We have used a cDNA probe complementary to induced virus RNA to quantify transcription of virus sequences in lymphocytes from mitogen-stimulated lymphocytes of the AKR, 129/J and Balb/c mice. Balb/c lymphocytes show the clearest case for induction of new virus sequences in response to stimulation. All strains including 129/J show expression of virus sequences in unstimulated control lymphocytes. The data indicate that mitogen induction of endogenous retrovirus is regulated at the transcriptional level.
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Further Studies on Anti-tumour Activity of Adenovirus Fibre Protein on Murine Sarcoma Tumours
More LessSUMMARYThe present study demonstrates that subcutaneous injection of purified adenovirus 12 fibre protein (FP) to 4-week-old Balb/c mice, 4, 2 and 1 day(s) before intramuscular injection of murine sarcoma virus (MSV-M) or 2 and 1 day before and 1 day after MSV-M injection causes significant inhibition of tumour growth (P < 0.001). The evidence suggests that treatment with FP leading to tumour inhibition is due to neither an interferon mechanism nor to inhibition of virus multiplication by FP in the reticuloendothelial system. It is postulated that immune functions, probably directed against MSV-induced cell antigens, might be the critical mechanisms underlying inhibition of tumour growth.
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Herpes Simplex Virus Infection in Human Monocyte Cultures: Dose-dependent Inhibition of Monocyte Differentiation Resulting in Abortive Infection
More LessSUMMARYMonocyte-enriched cultures of human blood leukocytes were exposed to herpes simplex virus (HSV) at different multiplicities of infection (m.o.i., from about 10 to 0.0001 p.f.u./cell). Highest maximum progeny virus titres were invariably obtained with low initial m.o.i., i.e. those between 0.01 and 0.0001 p.f.u./cell, while little if any infectious progeny was produced in cultures inoculated with the highest virus concentrations. By the time of maximum virus production, i.e. 5 to 7 days after inoculation, monocytes in the uninfected cultures had mostly differentiated to macrophages. This differentiation was partially inhibited in cultures initially exposed to the higher concentrations of HSV. Synthesis of HSV antigens was detected by indirect immunofluorescence both in the high m.o.i. cultures and in the productively infected cultures. By this criterion, a maximum of 10 to 15% of all adherent cells became infected in both culture types. It is suggested that the higher doses of HSV, by inhibiting cellular maturation, also prevent the subsequent completion of its own infectious cycle.
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Morphological Study of Virus-like Particles in Two Transplantable Tumours from BDX Rats
More LessSUMMARYVirus-like particles were found in two transplantable tumours, Sp56 and Sp6, from BDX rats. Sp56, a neurogenic sarcoma, contains abundant C-type particles in all stadia of morphogenesis. This tumour reacts with anti-Friend leukaemia virus gp70 and anti-Rauscher leukaemia virus p30 sera. Sp6, a fibrosarcoma, has abundant virus-like particles in the cytoplasm, very often associated with centrioles or basal bodies of a cilium. These particles consist of two concentric shells with a diam. of 60 to 65 nm. Released particles were found outside the cell with a diam. of 85 to 100 nm characterized by an envelope and an eccentrically located electron-dense nucleoid, surrounded by an intermediate layer. These virus-like particles show no cross-reaction with antisera against murine C- or B-type particles, but show ultrastructural similarity with virus particles recently described in Chinese hamster cells and in mouse cell lines infected with two retrovirus isolates from South-East Asian mice.
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Studies on an Interferon-sensitive Mutant of Mengovirus: Effects on Host RNA and Protein Syntheses
More LessSUMMARYInterferon induces an activity which strongly inhibits the growth of is-1, an interferon-response mutant of mengovirus. This activity is not expressed in protected cells infected with either is + (the wild-type parent) or vaccinia virus, or in cells infected with is-1 in the presence of actinomycin D. A failure of is-1 to shut off host RNA and/or protein synthesis could explain these observations. The present paper, however, shows that is-1 and is + are equally effective in suppressing host syntheses, and suggests that is + actively inhibits the interferon-mediated activity directed against is-1.
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Lack of Correlation between pp60 src Kinase Activity and Transformation Parameters in Cells Infected with Temperature-conditional Mutants of Rous Sarcoma Virus
More LessSUMMARYThe pp60 src kinase activity of cells infected with temperature-conditional mutants of Rous sarcoma virus (RSV), which induce only a partial transformation, was compared to the pattern of transformation parameters induced by these mutants. The tsGI251-infected cells were thermosensitive for hexose transport, fibrinolysis, morphological alterations and pp60 src kinase activity, but cold-sensitive for loss of growth control. The tsGI253-infected cells were similar to tsGI251-infected cells except that they were only slightly cold-sensitive for loss of growth control and had a very low pp60 src kinase activity which was temperature-insensitive. Thus, the pp60 src kinase activity measured in vitro with IgG as a substrate did not correlate in a consistent way with any of the measured parameters of transformation.
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Effect of Novobiocin and Other DNA Gyrase Inhibitors on Virus Replication and DNA Synthesis in Herpes Simplex Virus Type 1-infected BHK Cells
More LessSUMMARYThe four known inhibitors of the bacterial DNA gyrase (nalidixic acid, oxolinic acid, novobiocin and coumermycin A) were investigated with respect to their effect on the growth of uninfected BHK cells and the yield of virus from herpes simplex virus type 1 (HSV-1)-infected BHK cells. High concentrations of nalidixic acid and oxolinic acid (about 10 mm) were needed for 50% inhibition of cellular and viral multiplication with less than fourfold preferential inhibition of virus over cell growth. Novobiocin and coumermycin were effective at lower molar concentrations and the amount needed for 50% inhibition was 10-fold higher for cell growth than for virus yield. At 5 × 10−4 m, novobiocin inhibited DNA synthesis in uninfected cells to approx. 20% of non-treated controls, while virus DNA in infected cells was almost completely inhibited (approx. 1% of controls). Residual cellular DNA synthesis in infected cells was rather insensitive (approx. 90% of controls) to this concentration of novobiocin.
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Sequence and Antigenic Activity of the Region 93 to 113 of the Coat Protein of Strain U2 of Tobacco Mosaic Virus
More LessSUMMARYThe amino acid sequence of the coat protein of the U2 strain of tobacco mosaic virus (TMV) has been re-examined and completed. Four incorrect residue allocations in the published U2 sequence were identified. These were located in the region corresponding to residues 96 to 105. In addition, the identity of residues 106 to 112 was also determined. This latter region is of particular interest in antigenic studies, since the homologous region 108 to 112 in TMV (common strain) corresponds to an antigenic determinant of the depolymerized coat protein. Tryptic peptide 6 of strain U2 (residues 93 to 113) also contains an antigenic determinant, as shown by its ability to inhibit the reaction between U2 protein and specific antibodies.
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