- Volume 64, Issue 9, 1983
Volume 64, Issue 9, 1983
- Review Article
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- Bacterial
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Bacteriophage Distribution in Human Faeces: Continuous Survey of Healthy Subjects and Patients with Internal and Leukaemic Diseases
SUMMARYIn order to elucidate the ecological role of bacteriophages in the human intestine, we analysed the numbers of coliphages and of coliphage strains present in faecal samples collected from healthy individuals and from patients with certain intestinal diseases. The isolated phages were grouped according to their serological properties. The samples with low phage titres, observed in both healthy subjects and patients, contained mainly temperate phages (many were related to ø80 and λ), and those with higher titres, observed in patients, contained virulent phages. From successive surveys of coliphages and their host, Escherichia coli, in faecal samples of each subject, it was concluded that temperate phages are maintained in the human intestine through spontaneous induction of lysogenic bacteria. Qualitative and quantitative differences existed between phages isolated from faecal samples from healthy subjects and from patients. Simultaneous changes in the distribution patterns of coliphages and of the clinical symptoms were observed in a continuous survey of a leukaemic patient in a protective environmental ward.
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- Animal
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Purification and Partial Characterization of a New Enveloped RNA Virus (Berne Virus)
More LessSUMMARYIn Berne (Switzerland), a virus was isolated from a horse which was found to be serologically unrelated to known equine viruses. Its growth was unaffected by iododeoxyuridine and it was inactivated by organic solvents. A purification procedure involving ammonium sulphate precipitation and sucrose gradient equilibrium centrifugation was developed and viral activities were monitored using infectivity and enzyme-linked immunosorbent assays. Purified virions of density 1.16 g/ml were shown by negative staining electron microscopy to be roughly spherical and to measure 120 to 140 nm in diameter; projections (peplomers, about 20 nm long) were identified on the virion surface. In thin sections, an envelope and an elongated core structure could be distinguished. The core, measuring about 23 nm across and 104 nm in length, appears to assume a rod-, crescent- or open ring-shape within the envelope. It has a tubular structure and shows a transverse striation (periodicity 4.5 nm). Budding at the plasma membrane was observed. Berne virus is considered as a representative of a hitherto undefined family of widespread animal viruses serologically related to recent bovine isolates in Ames, Iowa (U.S.A.) and Lyon (France).
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Expression of Feline Infectious Peritonitis Coronavirus Antigens on the Surface of Feline Macrophage-like Cells
More LessSUMMARYGrowth of feline infectious peritonitis virus in a continuous feline cell line is described and evidence for the macrophage-like character of these cells is presented. Under one-step growth conditions, cytopathic changes and giant cell formation were observed 12 h after infection; more than 99% of the virus remained cell-associated 15 h after infection. Viral proteins at the surface of infected cells were detected by immunofluorescence. The exposed antigens were localized on four proteins with molecular weights of 225.5K, 175K, 138K and 25K using radioiodination followed by immunoprecipitation. Another viral polypeptide of 44K (the nucleocapsid protein) was only labelled when the cell membranes had been disrupted. Expression of viral antigens on the cell surface may be a significant factor in the immune pathogenesis of feline infectious peritonitis.
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Antibodies to Respiratory Syncytial Virus Polypeptides and their Significance in Human Infection
More LessSUMMARYThe human antibody response to respiratory syncytial (RS) virus infection was investigated using radioimmunoprecipitation analysis (RIPA). A total of nine RS virus-specific proteins, VP200, VGP95, VP68, VGP48, VPN41, VP35, VP27, VP23 and VGP20 were identified by comparing 35S- or 3H-labelled extracts of infected and uninfected HEp-2 cells, and by radioimmunoprecipitation using a hyperimmune human serum. Three glycopeptides, VGP95, VGP48 and VGP20, were identified by incorporation of [3H]glucosamine, and two of these (VGP48 and VGP20) were assumed to be part of a single disulphide-bonded polypeptide since they were precipitated by a monoclonal antibody raised against a surface protein. Human serum antibodies to three major RS virus proteins, VGP95, VGP48/VGP20 and VPN41 were measured by RIPA using radioiodinated RS virus antigens. Sera from a group of mothers whose babies escaped RS virus infection during a local epidemic showed increased antibody levels to VPN41 when compared to sera from mothers whose babies had become infected with RS virus within the first 6 months of life. In infants who remained uninfected with RS virus during the first 12 months of life the maternal gift of antibody decayed to about 50% at 3 months with traces of antibodies detected in a few sera at 12 months. The antibody levels detected in the sera of infants less than 3 months old convalescent from primary RS virus infection did not exceed the mean levels present in the serum of uninfected babies. Infants between the ages of 6 and 12 months were able to mount an IgG response to VPN41 and VGP48 but, unlike adults and older children, a particularly striking finding was their failure to produce antibodies to VGP95.
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The Molecular Biology of Rotaviruses. VI. RNA Species-specific Terminal Conservation in Rotaviruses
More LessSUMMARYThe use of T1 RNase fingerprinting of terminally labelled genomic double-stranded RNA species from various rotavirus isolates, to analyse the near terminal G-residue positions, has revealed an RNA species-specific fingerprint pattern covering approximately 40 nucleotides at the termini. These RNA species-specific terminal fingerprint patterns were found to be conserved in both rotavirus RNAs isolated from various animal species, and in isolates from a single animal species where gross divergence of internal RNA sequence for a particular RNA species was evident. This conservation of near terminal G-residue positions suggests that, internal to the short regions of absolute terminal sequence conservation that we have previously shown to be present on all rotavirus RNA species, there is a region of conserved sequence which is specific for a particular RNA species.
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Studies on the Structure and Organization of Avian Sarcoma Proviruses in the Rat XC Cell Line
More LessSUMMARYThe structure and arrangement of the multiple provirus copies of avian sarcoma virus in a rat XC cell line were studied by restriction endonucleases. The following observations were made: (i) the majority of the proviruses integrated randomly with respect to cell DNA; (ii) no gross deletions or rearrangements in the proviruses were observed; (iii) two types of proviruses (type I and type II) could be distinguished on the basis of restriction endonuclease cleavage sites; (iv) the virus rescued from these cells was derived from type II provirus, which has a novel EcoRI site between the env and pol genes; (v) most of the provirus units contained the src gene.
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Bovine Lymphosarcoma: Expression of BLV-related Proteins in Cultured Cells
More LessSUMMARYBovine leukaemia virus (BLV) is known to be the aetiological agent of enzootic bovine lymphosarcoma. As the mechanism of tumour induction is unknown, we analysed the viral proteins expressed in cultured bovine cells of different origin, i.e. from enzootic or sporadic tumourous tissues, normal cells infected or not with BLV, and the reference FLK-BLV cells. We also investigated BLVs of different origins. As well as the previously described BLV polyproteins in FLK-BLV cells (pr70 and pr45) we have also found two additional polyproteins, p52 and p27. In these four proteins we observed the combined antigenicities of p24, p15 and p12. We observed an additional polyprotein in p42, with the antigenicities of the p24 and the p15 in cells derived from tumour tissue or infected in vitro with naturally occurring BLV isolates. None of these BLV-coded proteins, nor others with cross-reacting antigenicities, were encountered in non-BLV-producing cultured cells from enzootic or sporadic tumours. The use of autologous sera did not permit the detection of any proteins in addition to the above gag-coded and env-coded proteins. Thus, there is no evidence for the existence of a gag-x fusion protein which could play a role in BLV-induced tumourigenicity.
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Baculovirus Replication: Glycosylation of Polypeptides Synthesized in Trichoplusia ni Nuclear Polyhedrosis Virus-infected Cells and the Effect of Tunicamycin
More LessSUMMARYInfected cell-specific polypeptides (ICSP) in Trichoplusia ni nuclear polyhedrosis virus (MNPV)-infected cells could be radiolabelled with various sugar precursors including mannose, N-acetyl glucosamine, N-acetyl mannosamine and glucose. Glycosylation occurred mainly late in infection. Eleven polypeptides were glycosylated including the major envelope protein, the polyhedron protein (very late in infection) and a major low molecular weight non-structural polypeptide (also very late in infection). Tunicamycin inhibited T. niMNPV replication and prevented the addition of N-acetyl glucosamine and, to a lesser extent, mannose to the appropriate proteins. This had the effect of enhancing the migration in SDS-polyacrylamide gels of some glycopolypeptides but not that of the polyhedron protein. Tunicamycin prevented the envelopment of nucleocapsids both within the nucleus and at the plasma membrane. Polyhedron formation did, however, occur in the presence of tunicamycin.
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Location and Orientation of Homologous Sequences in the Genomes of Five Herpesviruses
More LessSUMMARYMolecular hybridization experiments were carried out to investigate homologous regions in the genomes of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), equid herspesvirus 1 (EHV-1), pseudorabies virus (PRV) and varicella-zoster virus (VZV). Virion DNA probes from EHV-1, PRV and VZV hybridized to similar regions of the HSV genome, and the use of cloned DNA probes allowed heterologous genomes to be oriented with respect to homologous regions. The HSV-1 and HSV-2 genomes are colinear, the EHV-1 and VZV genomes are colinear with the IL or ISL genome arrangement of HSV, and the PRV genome is essentially colinear with the IL genome arrangement of HSV except that the region 0.1 to 0.4 fractional genome units appears to be inverted. A detailed analysis of sequences in the HSV-2 and PRV genomes to which the HSV-1 major capsid protein gene hybridized was carried out in order to demonstrate the application of molecular hybridization to the location of genes in heterologous genomes. The lesion in a DNA-positive temperature-sensitive mutant of PRV was mapped within the putative PRV major capsid protein gene. We conclude that the herpesviruses we have studied possess several highly conserved genes, and propose that they are similar in genetic organization despite presumably separate evolutionary histories.
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Processing of Glycoproteins Induced by Herpes Simplex Virus Type 1: Sulphation and Nature of the Oligosaccharide Linkages
More LessSUMMARYUsing the drug tunicamycin we have investigated the nature of the oligosaccharides on herpes simplex virus type 1 (HSV-1)-induced glycoproteins E and Y (gY is a newly identified glycoprotein which has the same apparent mol. wt. as gC but a more basic isoelectric point). Synthesis of both glycoproteins was inhibited by the drug, suggesting they contain N-linked oligosaccharides. Our finding, combined with the previous results of other workers, suggests that all the major HSV-induced glycoproteins have this type of carbohydrate modification. All of the major HSV-1-induced glycoproteins are modified by addition of inorganic sulphate. This modification occurs late in their maturation. Most inorganic sulphate appears to be attached to N-linked oligosaccharides but some is attached to other parts of glycoprotein E. Using HSV-1/HSV-2 intertypic recombinants, the mapping limits of that part of the glycoprotein E gene coding for differences in mobility between the two serotypes have been further narrowed and are located between coordinates 0.886 and 0.935.
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The Effect of Cycloheximide on the Accumulation and Stability of Functional α-mRNA in Cells Infected with Herpes Simplex Virus
More LessSUMMARYCells were infected with herpes simplex virus type 2, HSV-2(G), and incubated in the presence of cycloheximide (CX). When CX was removed and actinomycin D (Act D) added, α-polypeptides ICP 0 and ICP 4 were synthesized at low rates. If CX was removed without adding Act D, the rate of production of ICP 4 increased while that of ICP 0 remained constant. In cells treated with azetidine to enhance the production of ICP 4 and 0, accumulation of functional mRNA for ICP 4 (determined indirectly by translation in vivo) was reduced by concentrations of CX between 0.5 and 5.0 µg/ml, whereas mRNA for ICP 0 was unaffected by 50 µg/ml CX. CX apparently either inhibits the synthesis of ICP 4 mRNA or enhances its inactivation without affecting the production or degradation of ICP 0 mRNA. The accumulation of ICP 4 or ICP 0 mRNA of HSV-1(F) was unaffected by CX. The low levels of ICP 4 and ICP 0 mRNAs of HSV-2(G) that accumulated in the presence of CX disappeared rapidly after adding Act D, in contrast to those of HSV-1(F) which were stable. The ICP 4 mRNA of HSV-2(G) was stable, however, if made without CX or if in mixed infection with HSV-1(F) in the presence of CX. It is suggested that rapid inactivation may account for the low level of accumulation of functional ICP 4 and ICP 0 mRNAs of HSV-2(G) in the presence of CX, and that ICP 4 mRNA is protected by a protein made soon after normal infection. Such a protein may be carried in the virion of HSV-1(F).
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Delayed IgG-mediated Clearance of Herpes Simplex Virus Type 1 from the CNS but Not Footpad during the Early Stages of Infection: Possible Result of Relative Integrity of the Blood-Brain Barrier
More LessSUMMARYFollowing footpad inoculation in mice, herpes simplex virus type 1 spreads along nerves to the spinal cord where a myelitis causes hind limb paralysis beginning on day 6. Neutralizing antibody effectively prevents this illness only if given within 72 h. We therefore studied the timing of blood-brain barrier (BBB) disruption relative to the appearance of virus and inflammatory cell infiltrates in the spinal cord. Virus was detectable in dorsal root ganglion and spinal cord explants by 48 h. By 72 h, mononuclear cell infiltrates were evident in the spinal cord. By day 4, high titres of virus were demonstrable in the spinal cord. On day 6 125I-labelled IgG tracers penetrated the spinal cord BBB. In addition, using a passive transfer model, mice given neutralizing IgG completely cleared footpad virus within 72 h while brain virus titres were unaffected by IgG treatment up to day 7. These observations indicate that the BBB may prevent IgG-mediated virus clearance during the early stages of infection.
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Neuropathogenicity of Herpes Simplex Virus in Mice: Protection against Lethal Encephalitis by Co-infection with a Non-encephalitogenic Strain
More LessSUMMARYIntraperitoneal infection of susceptible mice with an apathogenic herpes simplex virus type 1 (HSV-1) strain prevented the lethal outcome of a challenge infection with a pathogenic strain, even if the challenge preceded the protective infection. It was found that the protective inoculation blocks the initial replication of the challenge virus. In addition, intraperitoneal infection with the protective HSV-1 strain led to the induction of a refractory state in the central nervous system, resulting in resistance to direct intracranial infection with HSV-1. This state is also inducible locally by intracerebral inoculation of a non-replicating mutant virus. The results indicate that HSV-1 strains differing in neurovirulence may differ in the induction or the sensitivity to this protective effect. Experiments with non-replicating HSV-1 temperaturesensitive strains demonstrated that protection against lethal infection does not depend on replication or expression of late genes of the protective strain. Inoculation of animals with detergent-soluble extracts of infected cells or infected and u.v.-irradiated syngeneic cells protected the animals against co-infection with encephalitogenic challenge virus. The experiments define this protective effect as an antigen-induced immediate host defence mechanism active within 24 h post-infection.
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Pathogenicity and Immunogenicity of Temperature-sensitive Mutants of Murine Cytomegalovirus
More LessSUMMARYTwo temperature-sensitive (ts) mutants (ts 21 and ts 24), which could complement each other, were isolated from cell culture-passaged murine cytomegalovirus (CC-MCMV). Characterization in vivo revealed that ts 21 lacked pathogenicity for suckling mice, neurovirulence for weanling mice and productive chronic infection in the salivary gland, whereas ts 24 was positive only for the first two parameters; both mutants retained immunogenicity comparable to that of the wild-type CC-MCMV. The immunogenicity of ts 21 and ts 24 was assessed by mortality of the immunized mice after challenge with virulent, salivary gland-passaged MCMV (SG-MCMV) and by replication of the challenge virus in the immunized hosts. The protective immunity conferred by ts 21 was maintained over a period of 3 months without production of infectious viruses in the salivary gland.
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Interrelation between Viral and Cellular DNA Synthesis in Mouse Cells Infected with the Parvovirus Minute Virus of Mice
More LessSUMMARYMouse fibroblasts arrested in G0 by isoleucine deprivation were inoculated with the autonomous parvovirus minute virus of mice (MVM). Infected cells were released from the G0 block by transfer to complete medium and their progression to and through the S phase was monitored. The onset of viral and cellular DNA synthesis coincided, suggesting that cellular factor(s) required for MVM DNA replication became available as soon as cells entered the S phase. Cellular DNA synthesis was reduced to about 60% by MVM infection. However, this inhibition did not decrease significantly the overall rate of DNA replication in infected cells because it was compensated by concomitant viral DNA synthesis. MVM infection delayed the movement of the cells out of S phase by at least 5 h. At any time post-infection, more than 95% of both viral and cellular DNA synthesis was sensitive to inhibition by aphidicolin. Since this drug is highly specific for cellular DNA polymerase α, the data are consistent with a major role of this enzyme in the in vivo DNA replication of autonomous parvovirus. The assembly of 95% of virus progeny particles was concomitant with a late phase of viral DNA replication which accounted for 30% of the total viral DNA synthesized. The inhibition of this residual viral DNA replication by aphidicolin reduced dramatically the size of the burst of infectious particles; this observation concurs with other evidence to suggest that encapsidation is driven by a late replication event sensitive to this drug.
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Inhibition by Interferon of Herpes Simplex Virus Thymidine Kinase and DNA Polymerase in Infected and Biochemically Transformed Cells
Amos Panet and Haya FalkSUMMARYThe induction of thymidine kinase (TK) and DNA polymerase was inhibited by interferon (IFN) in mouse L-cells infected with herpes simplex virus type 1 (HSV-1). The inhibitory activity of IFN at this early stage of HSV-1 replication was followed by a reduced virus yield and was dependent on the multiplicity of infection. The expression of a cloned thymidine kinase (tk) gene of HSV-1, in biochemically transformed L-cells (LTK+), was not affected by IFN. These same LTK+ cells, however, developed an antiviral state since, upon HSV-1 infection, the induction of TK and DNA polymerase of the replicating virus was inhibited by IFN. Furthermore, IFN inhibited the transactivation of the HSV-1 tk gene in the biochemically transformed LTK+ cells, which followed infection by a virus mutant defective in the tk gene (HSV-1 TK−). This transactivation is dependent on expression of immediate-early HSV-1 α-genes. These results indicate that IFN inhibits HSV-1 replication at an early step prior to DNA synthesis. In addition, IFN displays a differential effect on the HSV-1 thymidine kinase gene, either when part of the replicating virus or when expressed as a cellular gene in biochemically transformed cells.
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Hyporesponsiveness to Dog Interferon Induction in vitro
More LessSUMMARYSera from dogs that were hyporesponsive to interferon (IFN) induction blocked virus induction of IFN in spleen cells in vitro but not IFN action in dog kidney cells. Similarly, macrophages or their culture fluids were found to block IFN induction in lymphoid cells in vitro. Lymphoid cells deprived of phagocytic cells secreted IFN continuously for 4 days, whereas lymphoid cells in the presence of phagocytic cells produced IFN for only 1 day. It was speculated that the ‘hyporeactivity factor’ in the sera from hyporesponsive dogs was macrophage-derived.
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Sensitivity of Ovine Choroid Plexus Cells to Human and Other Animal Interferons
More LessSUMMARYThe sensitivity of ovine choroid plexus cells to the antiviral effects of the three major types of human and other animal interferons (IFNs) was tested. Ovine cells were found to be highly sensitive to the antiviral effects of natural human IFN-α and IFN-β but not IFN-γ. In addition, ovine cells were highly sensitive to recombinant DNA-derived human IFN-α2, IFN-β1 and IFN-γ. Furthermore, ovine cells were more sensitive (P < 0.01) to human IFN-αs than human fibroblasts (trisomic or disomic for chromosome 21) or bovine cells. Moreover, ovine cells were found to be highly sensitive to the three natural types of bovine, caprine, equine and porcine IFN preparations and relatively insensitive to mouse L-cell IFN.
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Interferon Production Associated with the Ageing of Primary Chick Embryo Cells Is Not Caused by Priming
More LessSUMMARYThe presence of a low level of interferon (IFN) activity was demonstrated in aged culture medium of primary chick embryo (CE) cells. The endogenous cytoplasmic level of 2′,5′-oligoadenylate (2′,5′-A) synthetase activity also increased during cell ageing. This increase was suppressed by addition of antiserum against chick IFN. In contrast, anti-chick IFN serum did not inhibit the ageing effect, i.e. the enhanced production of IFN upon induction after a prolonged preincubation of CE cells. This result indicates that the occurrence of the ageing effect is not mediated by the IFN system which had been expressed spontaneously.
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