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Volume 55,
Issue 1,
1981
Volume 55, Issue 1, 1981
- The Fifth Fleming Lecture
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Structural Analysis of Animal Virus Genomes
More LessPhilosophy of genome sequence studies. For the past four years I have been working on nucleotide sequence analyses of animal virus genetic material. During this time my attention has been more on the establishment and development of techniques than on the pursuit of analysis of one virus genome. Thus, I have worked on the genome RNAs and messenger RNAs of vesicular stomatitis virus and other rhabdoviruses, and also on the genome DNA of herpes simplex virus. It is evident that these form a rather disperse set of data on which to base this essay. Eventually, I decided to use the occasion to attempt to clear my own mind on the role of sequence analysis in research on animal viruses. I have from time to time encountered opinions hostile to or sceptical of such analysis. I have heard, often enough for it to stick, the term ‘mindless sequencing’.
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- Bacterial
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The Replication of Bacteriophage K DNA in Staphylococcus aureus
P. J. Rees and B. A. FrySummaryThree intracellular forms of K phage DNA were identified by sucrose gradient centrifugation and electron microscopy: (i) a form with sedimentation characteristics similar to mature phage DNA; (ii) a fast-sedimenting form (FSF) and (iii) a rapidly sedimenting complex (RSC). The FSF sedimented in a position intermediate between mature DNA and the RSC and comprised one genome length of phage DNA in association with a small mass of non-DNA material. Each RSC was associated with a discrete mass of non-DNA material about three times the size of that in the FSF and formed a large tangled structure containing up to 30 or more phage equivalents of DNA. The RSC was identified as the major replicative form.
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- Animal
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In vivo Effect of a New Mineral Condensed Ion (HPA 39) on Murine Friend Leukaemia
More LessSummaryHPA 39 is a tungsto-antimoniate compound, closely related to the mineral consensed ion HPA 23, from which it differs only by the presence of a potassium instead of a sodium ion inside the central cage. A single parenteral injection of HPA 39 on the same day as virus inoculation decreased the splenomegaly induced by Friend virus in DBA/2 mice and protected 90% of the infected animals against leukaemia. It also lowered the virus content in spleen extracts compared to untreated animals. The efficiency of treatment with HPA 39 on leukaemic mice at a late stage of the disease suggested that the compound may act at the cellular level as well as by inducing virus growth inhibition. HPA 39 also induced an early decrease of peripheral blood reticulocytes, and of the most differentiated erythroblasts in the bone marrow 1 day after injection of the compound. Mineral condensed ions therefore appear to have multiple biological effects both in vitro and in vivo.
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Experimental Infection of Inbred Mice with Herpes Simplex Virus
More LessSummaryThe peritoneal exudate cells (PEC) represent the first line of defence against the virus in the mouse model of intraperitoneal (i.p.) infection with herpes simplex virus (HSV). We have therefore studied interferon production and activation of natural killer (NK) cells in vitro in PEC of HSV-injected mice. Injection of HSV caused a marked increase in NK cell activity, as reported by others. PEC from HSV-injected mice also produced high titres of interferon. This observation may be important since induction of interferon appears to be the primary event whereas activation of NK cells — as generally accepted — represents a secondary effect of the interferon produced. The HSV-induced NK cells shared the properties of NK cells in that they were sensitive to a monoclonal anti-theta antibody and to a monoclonal anti-Qa 5 antibody. In contrast, the cells producing interferon were not sensitive to either antibody. Irradiation (200 R) of the mice 24 h before injection of the virus decreased interferon production by more than 90%. The identity of the interferon-producing cells is unknown, but they may represent B cells.
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Acute and Recurrent Herpes Simplex in Several Strains of Mice
More LessSummaryAcute and recurrent herpes simplex was studied after infection in the ear of two outbred and five inbred strains of mice. In all strains tested there was clinical evidence of infection, and a proportion of the mice became latently infected in the cervical ganglia. Six weeks after infection, when attempts were made to induce recurrent disease by stripping the ears of the mice with cellophane tape, a proportion of animals of each strain developed recurrent disease, characterized by erythema in the skin. At monthly intervals thereafter, the ears were stripped again and, on each occasion, a proportion of the animals developed recurrent disease, with the exception of Balb/c mice. The different reaction of Balb/c and other inbred strains might prove useful in studies on the mechanisms of control of recurrent herpes simplex.
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Molecular Heterogeneity of Hepatitis B Virus e Antigen in Liver and Serum
More LessSummaryHepatitis B virus e antigen (HBeAg) derived from liver at autopsy or from the serum of asymptomatic carriers has been characterized. The liver-derived HBeAg consisted of two different molecules, one with a mol. wt. of 30000 (monomer) and the other with a mol. wt. of 90000 (trimer), in a ratio of 3:1. Both were free of IgG. The serum-derived HBeAgs were heterogeneous with mol. wt. of 30000, 90000, 240000, 400000 and 540000. Among them, the so-called IgG-free HBeAgs consisted almost exclusively of the 30000 and 90000 molecular species, in a ratio of 1:9. The serum HBeAg of mol. wt. 90000 was further differentiated into two molecular species, one trimer and the other associated with albumin. The large mol. wt. HBeAgs (240000, 400000 and 540000) were associated with IgG in ratios of one molecule of HBeAg to one, two or three molecules of IgG respectively. The complete dissociation of the IgG molecule was not achieved by 5 m-urea treatment of such HBeAgs, suggesting that it was bound in an immune complex. A hypothetical model is proposed which describes the heterogeneity of the HBeAgs derived from both the liver and serum, and containing HBeAgs either in a free form or associated with serum IgG.
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The Inhibition of Vaccinia Virus Replication by 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB): an Effect at the Assembly Stage
More LessSummaryThe step sensitive to DRB (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole) in vaccinia virus replication has been investigated. Ninety µm-DRB extensively inhibited the yield of vaccinia virus after infection of Ehrlich ascites tumour cells. DRB did not inhibit cytoplasmic vaccinia DNA replication. Cytoplasmic viral RNA synthesis (both early and late) was also apparently unaffected and the virus RNAs thus synthesized were normal sized. The expression of early, intermediate and late proteins was not detectably impaired by DRB in vaccinia virus-infected cells. DRB inhibited vaccinia virus replication at the assembly stage since most of the virus DNA remained in a DNase-sensitive form in the infected cells and the virus was therefore not normally coated with virus proteins.
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Interference between Virulent and Avirulent Strains of Sendai Virus
More LessSummaryInterference between a virulent and an avirulent strain of Sendai virus was studied by plaque morphology and analysis of viral protein and RNA. On simultaneous infection a virulent, large plaque-forming strain (RL) was inhibited by an avirulent, small plaque-forming strain (RS). This interference was dose-dependent and decreased with u.v. irradiation of RS. One infectious particle was sufficient to induce the interference. SDS-polyacrylamide gel electrophoresis showed that interference was detectable in the synthesis of viral P protein; this was abolished when RS was u.v.-irradiated. Both growth and P protein synthesis of RL was restricted by superinfection with RS when this was done within 4 h after infection of RL, but the interference decreased gradually after this period and was not detectable after 8 h. Cycloheximide prolonged the period susceptible to superinfection by RS.
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Relation of Interferon Production to the Limited Replication of Newcastle Disease Virus in L Cells
More LessSummaryGrowth of Newcastle disease virus (NDV) in L cells, where the virus undergoes limited replication, has been compared to that in fully permissive BHK-21 host cells. The synthesis of viral proteins and the production of infectious progeny were found to occur normally at early times of infection in L cells. However, the subsequent amplification of viral protein synthesis did not take place and instead of infectious virions, non-infectious haemagglutinin was the predominant product. This shift of replication pattern from complete to incomplete virus production seemed to be temporally related to the appearance and accumulation of interferon (IFN) in the system. The addition of specific antiserum against mouse IFN to the infected L cell cultures was able to circumvent such restriction of virus growth. In the presence of the antiserum, synthesis of viral proteins was found to progress normally, with the production of a high amount of infectious progeny comparable to that of the permissive system. These results suggest that the limited replication of NDV in L cells may be due to interference by the endogenously produced IFN during the course of infection.
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Studies on the Restriction of Ecotropic Murine Retrovirus Replication in Mouse Teratocarcinoma Cells
More LessSummaryRetrovirus infection of cultured murine teratocarcinoma cells depends upon the state of differentiation. We have used two cell lines derived from a teratocarcinoma of mouse, strain 129. One, an undifferentiated pluripotential cell line (PCC4), is restrictive to viral infection, while the other, a differentiated myoblast-derived cell line (PCD1), is fully permissive to virus replication. We have shown that no virus RNA expression can be found in PCC4 cells 48 h post-infection and that no nucleic acid sequences can be found in an integrated form in PCC4 cells. However, the kinetics of formation of free proviral intermediates show that the three forms (I, II and III) of free virus DNA are synthesized in both PCC4 and PCD1. Free proviral DNA disappears gradually after 24 h in PCC4 cells while all forms increase in PCD1. These results suggest that the viral multiplication restriction occurs somewhere between the proviral DNA synthesis and integration of DNA in the cellular genome.
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The Detection of DNA Tumour Virus-specific RNA Sequences in Abnormal Human Cervical Biopsies by in situ Hybridization
SummaryWe have used the technique of in situ nucleic acid hybridization and autoradiography of thin frozen sections of human tissue to search for virus RNA sequences in human cervical tumours. Of cervical biopsies with abnormal cytology, 67% bound herpes simplex virus type 2 (HSV2) 3H-labelled DNA probes and 39% bound adenovirus type 2 (Ad2) 3H-labelled DNA probes, whereas control experiments with phage lambda 3H-labelled DNA probes, under the same conditions, bound to only 7% of cases. In contrast, normal cervical biopsies bound the three probes in only 23%, 17% and 8% of cases respectively.
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Characterization of Adenovirus Type 2 High Molecular Weight Nuclear Transcripts
More LessSummaryThe structure of adenovirus transcripts in the nuclei of infected cells has been studied. Evidence is presented for the existence in the steady-state population of nuclear RNA of two classes of adenovirus type 2 (Ad2)-specified large transcripts. The minor class consisted of molecules containing 3′ poly(A), 5′ cap structures and internal methylated nucleotides. Leader sequences at the 5′ end of these large poly(A)+ transcripts were identified and characterized by S1 hybridization analyses on alkaline agarose. They represented a spectrum of intermediates which ranged from completely unprocessed (5′ end at 16.5) to completely condensed (5′ end at 26.5) forms. S1 hybrid analyses located 3′ ends of large poly(A)+ nuclear RNA at 39, 50 and 61.5 on the Ad2 map. A fraction of these nuclear molecules consisted of transcripts that had undergone processing at both 3′ and 5′ ends of mRNA-coding regions since S1 bands corresponding to mature mRNAs for late proteins 52,55K, III, pVI and hexon, as well as 72K early protein were detected. The major class (75 to 85%) of large nuclear transcripts was not polyadenylated, but did contain capped leader sequences. These molecules yielded discrete bands, by S1 analysis, whose 3′ termini could be positioned at sites corresponding to 5′ ends of cytoplasmic mRNA (e.g. 47, 50, 51.5 and 66.5). The possible significance of these poly(A)-RNAs to the transcriptional programme of Ad2 is discussed.
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Polypeptides of Infectious Bronchitis Virus. I. Polypeptides of the Virion
B. Lomniczi and J. MorserSummaryInfectious bronchitis virus (IBV), strain Beaudette, grown in cultured cells contained five structural proteins with apparent mol. wt. of 170000 (p170), 94000 (gp94), 50000 (pp50) 30000 (gp30) and 26000 (p26). Both gp94 and gp30 are glycopeptides since they were labelled with [3H]glucosamine. The only phosphorylated polypeptide was pp50, and both it and gp94 were occasionally resolved into two bands. Two other polypeptides with mol. wt. of 28000 (p28) and 14000 (p14) were sometimes associated with the virus. In egg-grown virus two additional proteins were found with mol. wt. of 110000 (p110) and 75000 (gp75). The cell protein, actin, was also found in highly purified IBV virions. Different serotypes of either tissue culture-grown or egg-grown virus showed one of the two distinct polypeptide patterns of IBV described by Nagy & Lomniczi (1979) and Collins & Alexander (1980a, b). Strain Beaudette gave a pattern characteristic of the M type, while strain Connecticut gave a pattern characteristic of the C type. The polypeptides present in Connecticut virus were p170, gp98, pp50, gp28 and p26. Thus, the differences between the two patterns involve the mobility of both the large (gp94/gp98) and small (gp30/gp28) glycopeptides.
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Structural Characteristics of Nairoviruses (Genus Nairovirus, Bunyaviridae)
More LessSummaryViruses from six antigenic groups of arthropod-borne viruses [Crimean-Congo haemorrhagic fever (CCHF), Nairobi sheep disease (NSD), Qalyub (QYB), Sakhalin (SAK), Dera Ghazi Khan (DGK) and Hughes (HUG) serogroups], some previously categorized as bunyavirus-like viruses and others previously ungrouped, have recently been assembled by serological analyses into a new genus of viruses (Nairovirus genus) in the Bunyaviridae. Molecular studies of the virion RNA and viral polypeptides have been undertaken with representative members of the different serogroups [Hazara (HAZ) and Congo (CON) viruses, CCHF group; Dugbe (DUG) virus, NSD group; QYB, Omo and Bandia (BDA) viruses, QYB group; Avalon (AVA) virus, SAK group; DGK and Abu Mina (AM) viruses, DGK group; and HUG virus, HUG group]. In agreement with a recent study of QYB virus and in part agreement with an earlier report on DUG virus, the results of these molecular analyses indicate that nairoviruses have: (i) three virion RNA species (large, L, medium, M, and small, S) with apparent mol. wt. of 4.1 × 106 to 4.9 × 106, 1.5 × 106 to 1.9 × 106 and 0.6 × 106 to 0.7 × 106 respectively; (ii) a 48 × 103 to 54 × 103 mol. wt. nucleocapsid (N) polypeptide; and (iii) two external glycopolypeptides, 72 × 103 to 84 × 103 mol. wt. (G1) and 30 × 103 to 40 × 103 mol. wt. (G2). Cross-immune precipitation analyses have confirmed that viruses in the Nairovirus genus share antigenic determinants and are antigenically distinct from representative members of the Bunyavirus, Phlebovirus and Uukuvirus genera (Bunyaviridae).
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One Functional Copy of the Long Terminal Repeat Gene Specifying the Immediate-early Polypeptide IE 110 Suffices for a Productive Infection of Human Foetal Lung Cells by Herpes Simplex Virus
More LessSummaryThe HSV-1/HSV-2 intertypic recombinant Bx1 (28-1) is heterotypic for the repeat sequences flanking the long unique region of the genome (IRL and TRL) and expresses both the immediate-early polypeptide IE 110 of HSV-1 and its functionally equivalent polypeptide IE 118 of HSV-2. The genome structures of five subclones of this recombinant and the immediate-early polypeptides they induce have been analysed. Subclone 14 lacked most of the IRL sequence, including the region from which part of the mRNA for IE 110 is transcribed, and expressed only the HSV-2 IE 118. Subclone 22 lacked almost all of TRL including the gene for IE 118 and induced only the HSV-1 IE 110. Since both subclones produced viable progeny in HFL cells it follows that expression of only one copy of the equivalent genes in TRL and IRL, here coding for the distinguishable polypeptides IE 110 or IE 118, is sufficient for successive complete cycles of virus replication.
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A Comparison of the Genomes of Polioviruses by cDNA:RNA Hybridization
More LessSummaryRNAs from 11 strains of the three serotypes of poliovirus were isolated from infected HeLa cells and hybridized with representative, radioactive complementary DNA (cDNA) from either poliovirus type 1 or type 2. Hybridization and thermal denaturation were analysed by hydroxyapatite chromatography. cDNA to either poliovirus type 1 or 2 is competent to detect about 70 to 80% of the sequences in RNAs from heterotypic strains and more than 92% of the RNA sequences in homotypic strains. The three polio serotype genomes appear to have diverged from each other by about 20%. Both poliovirus type 1 and type 2 representative cDNA can be used for the detection of poliovirus genomic sequences in RNA isolated from tissue.
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The Plaque-forming Factor for Mink Lung Cells Present in Cytomegalovirus and Herpes-Zoster Virus Stocks Identified as Mycoplasma hyorhinis
More LessSummaryPrevious investigation of the ability of cytomegalovirus and varicella-zoster virus to replicate in a variety of cell lines suggested that both virus types plaqued with high efficiency in mink lung cells. However, many of the virus isolates used appeared to be contaminated with mycoplasma. We now report that the observed cytopathic effect is due to a mycoplasma which grows lytically to high titre in mink lung cells, but is difficult to cultivate in cell-free media. The mycoplasma was plaque-purified and shown to contain DNA with a buoyant density of 1.684 g/ml, with restriction endonuclease patterns identical to the porcine mycoplasma M. hyorhinis. This was confirmed by serological identification.
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Restriction Endonuclease Analysis of the DNA from Varicella-Zoster Virus: Stability of the DNA after Passage in vitro
More LessSummaryDNA was extracted from nucleocapsids isolated from WI-38 cells infected with two different strains of varicella-zoster virus (VZV). The DNAs were treated with each of six restriction endonucleases (EcoRI, HindIII, BglI, BglII, Sal I and BamHI) and small, but reproducible differences in restriction endonuclease patterns were observed. These strains were passaged in WI-38 cells and in primary guinea-pig embryo (GPE) cells, followed by restriction endonuclease analysis of the DNAs. No changes were observed in the restriction profile of the DNA of one of the strains (VZV-KMcC) after 46 passages in WI-38 cells but small differences were observed after 72 passages. No changes were observed after 30 passages of another strain (VZV-AW) in WI-38 cells. Twenty passages of VZB-KMcC in GPE cells did result in minor alterations of its DNA. It was concluded that VZV DNA was sufficiently stable after multiple passages in WI-38 cells to make restriction endonuclease analysis a valuable epidemiological tool for strain differentiation.
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Polypeptide Synthesis Catalysed by Components of Pichinde Virus Disrupted by Detergent
More LessSummaryPichinde virus preparations were investigated for ribosomal components and associated activities. After detergent treatment and fractionation, viral components were assayed for polypeptide synthesis, elongation of nascent polypeptide chains, and mRNA activity. It was demonstrated that these subviral particles could synthesize polypeptides when exogenous mRNA template, aminoacyl-tRNAs, translation factors, GTP and appropriate cations were added. Undisrupted, whole virions could not synthesize polypeptide; disruption and subsequent centrifugation of subviral ribosomes was a prerequisite for the biosynthetic activity described.
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Oligonucleotide Fingerprint Analysis of Tacaribe Virion RNA
More LessSummaryPolyacrylamide gel electrophoretic analysis of RNA segments of the arenaviruses Pichinde (Pic) and Tacaribe (Tac) showed them to be distinguishable in that Pic S RNA had a slower electrophoretic mobility than Tac S RNA. The L and S RNA segments of Tac virions were found to have distinct RNase T1 oligonucleotide fingerprints, indicating that they are unique RNA species. The oligonucleotide patterns of the Tac L and S RNAs were also distinct from those of the corresponding Pic RNA segments.
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