- Volume 68, Issue 2, 1987
Volume 68, Issue 2, 1987
- Bacterial
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Controlling Mechanisms for Expression of the Bacteriophage T4 β-Glucosyltransferase Gene
More LessSUMMARYThe plasmid pTHF3047 carries a 2200 bp fragment containing the phage T4 β-glucosyltransferase (βgt) gene and part of the upstream gene 42 cloned in pBR313 under the control of the amp promoter PI. In T4-infected cells the βgt gene may be expressed by a mechanism antagonizing Rho action. The plasmid pTHF3047 expressed about threefold higher β-glucosyltransferase activity in a strain carrying the polarity-suppressing rho-102 mutation than in an otherwise isogenic rho + strain, and production of T4 agt−βgr− phage was strongly stimulated. The plasmid copy number and the total T4-specific transcription was the same in the two strains. Two T4-specific transcripts from the plasmid, 600 bases and 1850 bases, were identified by Northern hybridization. Comparison with the T4 and plasmid maps suggested that both transcripts were initiated at PI, the 600 base transcript ending at the Rho-dependent terminator t42 between gene 42 and βgt, and the 1850 base transcript reading through this terminator to the end of the jlgt gene. This analysis places t42 at position 25·1 on the T4 map, and a Rho-independent βgt terminator at position 23·8. The three-fold higher βgl expression in the rho− strain may be partially accounted for by Rho control of transcription. In the rho+ strain about half of the transcripts stopped at t42, while in the rho− strain readthrough appeared slightly higher. Thus, the t42 terminator was observed also on the plasmid, but appeared considerably less effective there than in the phage DNA in infected cells.
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Abortive Infection by Bacteriophage Me1 of Escherichia coli K12 Strains Bearing the Plasmid ColV, I-K94
More LessSUMMARYBacteriophage Me1 is unable to grow on Escherichia coli strains harbouring the ColV,I-K94 plasmid. The nature of this inhibition was investigated, and it was found not to be due to restriction, superinfection exclusion or receptor-mediated resistance, but to be a new example of plasmid-mediated abortive infection. Investigation of events occurring during abortive Me1 infection revealed some differences from previously described cases, especially with regard to late protein synthesis, which did occur, albeit showing abnormal amounts of some proteins. No major differences were observed in membrane permeability of productively and abortively infected cells. Phage-directed DNA synthesis was reduced in abortively infected cells. Comparative studies of Me1 and T4 revealed a striking similarity despite some minor differences.
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Electron Microscopic Study of the Process of DNA Ejection from the Head of PL-1, a Lactobacillus casei phage
More LessSUMMARYThe process of DNA ejection from the head of PL-1, a Lactobacillus casei ATCC 27092 phage, was studied by electron microscopy by counting the number of ghost particles with empty heads among phages already adsorbed to the cell. The process of DNA ejection was temperature- and live cell-dependent.
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- Animal
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Antigenicity and Immunogenicity of Synthetic Peptides of Foot-and-Mouth Disease Virus
More LessSUMMARYPeptides reactive with two neutralizing monoclonal antibodies raised against intact foot-and-mouth disease virus A10 were identified with the aid of all overlapping (hexa)peptides of the outer structural viral protein VP1 and located on the viral surface. Using this procedure, it was possible to define those amino acids within a peptide which were critical in the binding of antibody to that peptide. One eight amino acid long peptide, containing six such amino acids, was virtually indistinguishable from viral antigen in its ability to bind monoclonal antibody as determined by competition tests. Another peptide, which was able to induce neutralizing activity as well, showed no competition and possessed fewer amino acids contributing to binding. This peptide appeared to be an incomplete epitope. Comparison of our data with those of others suggests that this may apply commonly to the reactive peptides described.
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A Neutralizing Epitope on Human Rhinovirus Type 2 Includes Amino Acid Residues between 153 and 164 of Virus Capsid Protein VP2
SUMMARYUse has been made of a monoclonal antibody (designated 8F5) to map a neutralizing epitope on the viral capsid protein VP2 of human rhinovirus 2 (HRV2). This antibody which was raised against the native virus, neutralizes HRV2 and is also capable of recognizing denatured VP2 on Western blots. To examine the binding site of 8F5, VP2 of HRV2 was expressed in Escherichia coli. Deletions starting at the 3′ end were then introduced into the gene for VP2 using Bal-31 nuclease. Polypeptides shortened at the carboxy terminus of VP2 were obtained from the deletions and were blotted onto nitrocellulose. The samples were then probed with monoclonal antibody 8F5. Recognition by 8F5 was maintained as long as the expressed polypeptide contained the VP2 sequence up to amino acid 164 or beyond. However, when the VP2 sequence was truncated to amino acid 153 or less 8F5 was no longer able to bind. The neutralization epitope (or part of it) recognized by 8F5 on VP2 is therefore located between amino acids 153 and 164.
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Effects of Extracellular Cations on Translation in Poliovirus-infected Cells
More LessSUMMARYThe effect of pH and different concentrations of added monovalent and divalent cations on translation in poliovirus-infected HeLa cells has been examined. A strong effect on protein synthesis was observed when the concentration of sodium ions was modified. If cells were placed in a hypotonic medium after virus adsorption, no shut-off of cellular translation took place, nor were viral proteins synthesized. An increase in the multiplicity of infection partially overcame this effect. Reversal of the shut-off of cellular translation and inhibition of viral protein synthesis was achieved when cells were placed in hypotonic medium 2 h after infection. Modification of divalent cations (calcium or magnesium) had little or no effect on the pattern of translation. On the other hand, acidic pH (below 6) inhibited both cellular and viral protein synthesis, whereas basic pH had no influence. During infection the synthesis of poliovirus proteins reached a maximum at about 4 to 6 h and then declined. This inhibition of viral translation was partially prevented if cells were placed in a medium containing a high concentration of potassium although the cytopathic effect was prevented. These results indicate that viral protein synthesis and the cytopathic effect were, to a large extent, influenced by the external monovalent ion concentration.
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Cation Content in Poliovirus-infected HeLa Cells
More LessSUMMARYPoliovirus-infected HeLa cells increased their permeability to monovalent ions from the third hour post-infection. At that time infected cells lost their content of potassium ions as measured by efflux of the potassium analogue 86Rb+. The rate of release of 86Rb+ from cells increased as infection proceeded, and was not sensitive to ouabain or quinidine, inhibitors of the Na+/K+ ATPase and Ca2+-induced K+ release system, respectively. The leakage of 86Rb+ was only slightly sensitive to furosemide, an inhibitor of the Na+/K+/Cl− cotransport system, suggesting that the mechanism of release involved an increased passive permeability of the cell membrane. The calcium content or its efflux did not vary significantly in the infected cells. Neither were there alterations in the intracellular pH throughout infection.
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Molecular Epidemiology of Infectious Bronchitis Virus in The Netherlands
SUMMARYTwelve Dutch isolates and the M41 strain of infectious bronchitis virus (IBV), a coronavirus of chickens, were characterized by cross-neutralization and T1 fingerprinting to elucidate their evolutionary relationship. The T1 fingerprinting showed that the Dutch isolates formed two clusters. The first cluster contained strains H52, H120, D387, V1259, V1385 and V1397; the estimated sequence homology is 99%. Cluster two comprised strains D207, D274, D212, D1466, D3128 and D3896, which have about 95% sequence homology. The M41 virus did not belong to either cluster. The four different serotypes which arose in the late 1970s belonged to cluster two and appeared to be different from the vaccine strains (H52 and H120) used at that time. This indicates that the strains were newly introduced and could have arisen from a common virus. On the other hand, three recently isolated field strains were genetically closely related to the vaccine strains H120 and H52 (cluster one), suggesting that these live vaccine strains themselves could have given rise to these serologically altered field isolates. The data are relevant to the development of new vaccine strategies.
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Morphological and Biochemical Properties of Four Members of a Novel Group of Reoviruses Isolated from Aquatic Animals
More LessSUMMARYThe morphological, biochemical and growth characteristics of four members of the Reoviridae, three from the fish hosts, golden shiner (Notemigonus crysoleucas), chum salmon (Oncorhynchus keta) and channel catfish (Ictalurus punctatus) and one from American oyster (Crassostrea virginica), were compared. Electron microscopy of negatively stained virions revealed icosahedral particles approximately 75 nm in diameter composed of a double capsid. Complete particles had buoyant densities in CsCl of 1·34 to 1·36 g/ml. The viruses replicated well in several fish cell lines, forming plaque-like syncytia in monolayer cultures. Each virus could be distinguished by the range of cell lines supporting its growth. Polyacrylamide gel electrophoresis showed that the genome of each virus was composed of 11 segments of dsRNA distributed among three size classes. There were three large, three medium and five small segments in each genome and each isolate had a unique electropherotype. The segments ranged from 2·5 × 106 to 0·31 × 106 mol. wt. with a total genome of approximately 15 × 106 mol. wt. Analysis by SDS-PAGE revealed that each virus had five major structural proteins. There were two large polypeptides of approximately 135000 and 125000 mol. wt., one medium size polypeptide of 70000 mol. wt. and two small polypeptides of 45000 and 34000 mol. wt. Of the major structural proteins, those of approximately 70000 and 34000 mol. wt. were consistently present in the highest concentrations. Minor virion proteins were detected but were not characterized. These four viruses, isolated from aquatic animals, were unlike viruses of the six established genera of the Reoviridae.
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Characterization of Novel Viral Polyproteins Detected in Cells Infected by the Flavivirus Kunjin and Radiolabelled in the Presence of the Leucine Analogue Hydroxyleucine
More LessSUMMARYVero cells were infected by Kunjin virus and radiolabelled in the presence of the leucine analogue, threo-β-hydroxy-dl-leucine (THL). This analogue is known to prevent preprotein processing in cell-free systems when incorporated into signal peptides. Novel Kunjin virus-specified proteins were detected, namely gpl40, pl20 and gp92; the designations for the proteins indicate their approximate M r × 10−3 and whether they are glycosylated. The glycoproteins gp 140 and gp92 were observed in cells labelled with [3H]mannose and bound to concanavalin A. Limited proteolytic digestion of gpl40, gp92 and gp66 (related to the envelope protein E), and tryptic peptide mapping of these three glycoproteins and p120 indicated that all four were closely related. The glycoproteins gpl40 and gp92 were also detected in cells infected by West Nile virus radiolabelled in the presence of THL. Other effects of THL in Kunjin virus- infected cells were (i) a reduction in the incorporation of radioactive amino acids or mannose, (ii) a decrease in the yield of haemagglutinin and infectious virus, (iii) an inhibition of processing of gp66 to gp53 and (iv) an apparent inhibition of synthesis of P98 and P71. We suggest possible explanations for the THL-induced changes in infected cells based on recent models proposed for the synthesis of the yellow fever and West Nile viral proteins.
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Semliki Forest Virus-induced, Immune-mediated Demyelination: Adoptive Transfer Studies and Viral Persistence in Nude Mice
More LessSUMMARYAdoptive transfer experiments in athymic nude mice demonstrated that the demyelination seen in the central nervous system (CNS) following Semliki Forest virus (SFV) infection was directly dependent upon sensitized T lymphocytes. Antibodies generated during the infection did not seem to be involved in the demyelination, but thymus-dependent antibodies (IgG) were responsible for the reduction of brain virus titres. In the absence of a T cell response and T cell-dependent antibody production, virus persisted in the CNS for several months. Despite persistence of high virus titres for this time, only mice eventually developing a CNS inflammatory response developed lesions of demyelination. In the absence of an inflammatory response no demyelination was apparent even after several months of persistent infection. Administration of anti- SFV hyperimmune serum intracerebrally to both infected and control mice did not produce demyelination but resulted in CNS tissue degeneration with marked pycnosis.
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Entry Pathway of Vesicular Stomatitis Virus into Different Host Cells
More LessSUMMARYA biochemical and morphological investigation of the mechanism of entry of vesicular stomatitis virus (VSV) into host cells of mammalian (HeLa), avian (CER), piscine (EPC) and arthropod (Aedes albopictus) origin, is described. VSV was capable of infecting all cell lines tested by a endosome- and/or a lysosome-dependent step since ammonium chloride and amantadine blocked the early stages of infection. Complement-dependent immune lysis of infected host cells provided evidence that in none of the four different cell types examined did insertion of VSV antigens occur from the outside to any great extent on the cell surface. When the entry process was studied by electron microscopy, virus particles were seen to be bound to the cell surface at 0 °C. After warming at 37 °C for homeothermic cells or at 26 °C for poikilothermic cells, virus was detected within coated pits and coated vesicles and, later, in lysosomes. VSV entry was seen to take place by endocytosis in all four cell lines, which were derived from phylogenetically unrelated species.
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Genetic Control of Scrapie: Incubation Period and Plaque Formation in I Mice
More LessSUMMARYThe host component of control of scrapie incubation period in the mouse is manifested largely through the action of the Sine gene. Only one mouse strain (VM) has been found that is p7p7 (prolonged incubation for ME7 agent) and two other strains have been derived from VM. All other strains, designated s7s7, have a short incubation for ME7. In the present study, the I strain was shown to fulfil the criteria that are characteristic of mouse strains with the p7 allele of Sine: (i) a comparatively long incubation period for ME7 and a short incubation period for 22A, (ii) the incubation period for F, hybrid mice (s7s7 x p7p7) either fell between the incubation periods for the parental strains (with ME7) or were longer than either parent (with 139A and 22A), (iii) amyloid plaques occurred following injection of ME7 and 87V but not after 22A or 139A, (iv) lesion profiles for four scrapie strains were similar in I mice and p7p7 mouse strains, and (v) injection of 87V led to disease in less than 300 days. Finally, allelism tests using F, hybrid mice (I × a p7p7 mouse strain) and progeny of backcrosses between these F, mice and I mice failed to reveal the segregation of additional major genes affecting scrapie incubation period.
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Glycoproteins of Human Parainfluenza Virus Type 3: Affinity Purification, Antigenic Characterization and Reconstitution into Lipid Vesicles
More LessSUMMARYMonoclonal antibodies to the envelope glycoproteins, HN and F, of human parainfluenza virus type 3 were coupled to a Sepharose 4B matrix and used for affinity purification of the viral glycoproteins. The purity of the glycoproteins was demonstrated by SDS-PAGE followed by fluorography or silver staining. The antigenicity of the glycoproteins was determined by immunization of rabbits; polyclonal rabbit antisera demonstrated inhibition of functional activities of the virus glycoproteins. The F glycoprotein, when reconstituted into lipid vesicles, showed distinct spike-like projections similar to those of intact virions.
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Mechanism of Protection During the Early Phase of a Generalized Viral Infection. II. Contribution of Polymorphonuclear Leukocytes to Protection against Intravenous Infection with Influenza Virus
More LessSUMMARYThe contribution of phagocytes to the early protection of mice inoculated intravenously with influenza virus was investigated in phagocyte-depleted mice. Following the inoculation of a sublethal dose of influenza virus, virus titres in the liver and lung of both untreated and carrageenan-treated mice were reduced rapidly by day 1 and decreased slowly to reach an undetectable level by day 7. The titres in γ-irradiated mice decreased transiently by day 1 and increased progressively thereafter to kill all of the hosts by day 8. The clearance of virus from blood at the early stage of infection was retarded by γ-irradiation but not by carrageenan treatment. In addition, increase in virus titres in the liver and lung in the early stage of the infection was prevented by adoptive transfer with syngeneic polymorphonuclear leukocytes into γ-irradiated mice. No significant rise of neutralizing antibody was detectable by day 3 after the inoculation, in any of the three groups of mice. These observations imply that γ-sensitive and carrageenan-resistant polymorphonuclear leukocytes play a protective role at the early stage in the infection, whereas fixed macrophages or natural killer cells, both of which are carrageenan-sensitive and γ-resistant, scarcely participate in the early phase.
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Protective Mechanisms against Pulmonary Infection with Influenza Virus. I. Relative Contribution of Polymorphonuclear Leukocytes and of Alveolar Macrophages to Protection during the Early Phase of Intranasal Infection
More LessSUMMARYThe relative contribution of polymorphonuclear leukocytes and macrophages in the early protection against intranasal infection of mice with influenza virus was investigated. Virus multiplication in the lung in the early phase of infection with less than 1·5 × 103 plaque-forming units was enhanced by X-ray irradiation. The intranasal administration of carrageenan did not influence the titre of virus. However, when mice were infected with 1·5 × 104 plaque-forming units, the virus titre was elevated by intranasal administration of carrageenan as well as by X-ray irradiation, but not by intraperitoneal administration of carrageenan. The intranasal administration of carrageenan not only inhibited the phagocytic activity of alveolar macrophages but also enhanced susceptibility to the virus. On the other hand, polymorphonuclear leukocytes were capable of phagocytosing the virus in vitro and were non-permissive for virus infection. Neutralizing antibody and interferon were not detectable in the early stage of the infection. These results suggested that polymorphonuclear leukocytes (X- ray-sensitive, carrageenan-resistant) were the cells primarily responsible for early protection in influenza virus infection and that after infection with a high dose of the virus alveolar macrophages (X-ray-resistant, carrageenan-sensitive) also played a protective role in the early phase.
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Purified Influenza Virus Nucleoprotein Protects Mice from Lethal Infection
More LessSUMMARYLocal administration of nucleoprotein purified from X31 (H3N2) influenza A virus primed for A virus cross-reactive cytotoxic T cells and resulted in substantial protection (75 %) of mice from a lethal challenge with the heterologous mouse-adapted A/PR/8/34 (H1N1) virus. By following the course of a lethal virus challenge we found that nucleoprotein priming did not prevent virus infection but rather aided recovery. Nucleoprotein-primed mice suffered initial symptoms of infection, i.e. weight loss and surface temperature changes, but started to recover after approximately 7 days. We suggest that such heterotypic protection can be attributed to priming of A virus crossreactive cytotoxic T cells.
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Demonstration of an Immunosuppressive Action of Detergent-disrupted Influenza Virus on the Antibody Response to Inactivated Whole Virus Vaccine
More LessSUMMARYIn a series of experiments performed in hamsters and mice, administration of mixtures of detergent-disrupted (SV) influenza A X49 (H3N2) virus and inactivated X49 whole virus (WV) vaccine induced lower serum antibody titres than equivalent or lower doses of WV vaccine alone. This reduction in antibody titre was also observed using influenza A (H1N1) and influenza B (B/Hong Kong/8/73) SV and WV vaccine preparations. The results suggested that SV preparations can suppress the serum antibody response to WV vaccine. A suppressive effect of SV influenza virus on WV vaccine was also observed in an in vitro antibody-forming system, using primed mouse spleen cells. In this system, SV induced markedly lower IgG and IgM antibody responses than WV vaccine, and mixtures of SV with WV reproducibly resulted in lowered antibody responses compared to those elicited by WV alone. Possible reasons for these findings are discussed in the light of the known low immunogenicity observed for split and subunit influenza virus vaccine preparations in animals and in unprimed human populations.
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Human Papillomavirus Type 16 DNA from a Vulvar Carcinoma in situ is present as head-to-Tail Dimeric Episomes with a Deletion in the Non-coding Region
More LessSUMMARYA number of genital cancer biopsy samples were screened for the presence of human papillomavirus type 16 (HPV-16) DNA sequences. One of these samples (a vulvar carcinoma in situ) was found to contain more than 100 copies of HPV-16 DNA sequences per cell. Using this tumour DNA, a genomic library was constructed in bacteriophage lambda and the library was screened for recombinant phage containing HPV-16 sequences. Five recombinant phage clones were isolated and their DNA was analysed by restriction endonuclease digestion and blot hybridization. All five recombinants contained two copies of the HPV-16 genome present in a head-to-tail arrangement. The data are consistent with the presence of HPV-16 sequences in the tumour DNA arranged as genomic dimers in a circular episomal configuration. The HPV-16 genomes contained a deletion within the non-coding region, a region which includes the viral origin of DNA replication and transcriptional control sequences. Possible consequences of this deletion for viral replication and transcription are discussed.
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Virus-Lymphocyte Interactions during the Course of Immunosuppressive Virus Infection
More LessSUMMARYMalignant rabbit fibroma virus (MV) is a lymphocytotropic leporipoxvirus which produces profound immunological dysfunction and lethal fibromyxosarcoma. We examined virus recovery from splenic lymphocytes as a function of time after inoculation in vivo, and correlated this with both immunological function and expression of virus-induced host suppressor activity. MV was most abundant in lymphocytes obtained 4 days following inoculation. At that time, immune function was relatively normal and host suppressor activity was not observed. By 7 days after infection, when active host immunosuppressor functions were observed, virus recovery was decreased. Eleven days post-inoculation host immune function began to recover despite increasing virus-induced tumours and developing opportunistic infection. Simultaneously, MV was no longer recoverable from spleen cells. Spleen cells from day 11 tumour-bearing rabbits did not support MV replication as efficiently as did normal or day 4 or 7 splenic lymphocytes, but they did not alter the ability of MV to grow in the latter cells. By fluorescence examination and cytofluorography, splenic lymphocytes bearing MV antigens were abundant 7 days after infection but disappeared by 11 days. This was temporally related to production of neutralizing antibody to MV, and development of virus-specific lymphocyte proliferative activity. The composition of splenic lymphocytes changed as well: the normal ratio of about 1:1 for B and T cells changed to 1:2 by day 7, and then inverted to almost 2 :1 by day 11. Rabbits infected with MV thus appear to recover their immune function, concurrently eliminate virus-infected lymphocytes, and elaborate high titres of neutralizing serum antibodies despite progressive infections and tumour development.
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Proteins Antigenically Related to Peptides Encoded by the Mouse Mammary Tumour Virus Long Terminal Repeat Sequence Are Associated with Intracytoplasmic A Particles
More LessSUMMARYIntracytoplasmic A particles (CAP), previously identified as cytoplasmic nucleo-capsid precursors to mouse mammary tumour virus (MMTV), reacted strongly in immunodiffusion tests with polyclonal antibodies raised against synthetic oligopeptides derived from the open reading frame (ORF) in the long terminal repeat (LTR) of MMTV. In Western blots, several CAP proteins (p80, p72–68, p36, p32, p 18–12) were reactive with polyclonal antibodies raised against three separate LTR ORF synthetic peptides. Disrupted MMTV virions did not react with the anti-LTR ORF peptides suggesting that ORF proteins were excluded from mature virions during maturation. Serial dilution of anti-LTR ORF antibody demonstrated that the most reactive CAP proteins in Western blots migrated as a doublet band with estimated molecular weights of 68000 and 72000. Reactivity of anti-LTR ORF serum with these and other CAP proteins was removed upon preincubation with free synthetic peptide. Absorption with LTR synthetic peptides did not affect the reactivity of antibodies directed against MMTV gag proteins with similarly sized CAP polyproteins. LTR ORF-related proteins with molecular weights similar to those associated with CAP were also detectable in Western blots of total cytoplasmic extracts of MMTV-infected mammary tumour cells.
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Differences in the pI Heterogeneity of Virion and Intracellular Moloney Murine Leukaemia Virus p30s
More LessSUMMARYAt least three different p30 forms which vary in isoelectric point (pI) were previously shown by two-dimensional (2D) gel electrophoresis to be present in purified virions obtained from several strains of murine leukaemia virus (MLV). This heterogeneity which had been identified by Coomassie Brilliant Blue staining has been further characterized by immunological techniques. Using as substrates two Moloney (M) MLV chronically infected cell lines (M JD-54 and clone 2 cells), we found that (i) all p30s had the antigenicity of M-MLV p30, when analysed by immunoblotting of virion proteins with anti-p30 sera, and (ii) when cells were labelled with p5S]methionine, a 14C-amino acid mixture, or [l4C]serine and lysates of purified virions were immunoprecipitated with goat anti-p30 sera, four p30 spots (pI 6·0, 6·1, 6·3 and 6·6) could be clearly identified. These results strongly support the viral origin of the heterogeneous p30 spots. We next examined infected cell lysates in an attempt to pinpoint the molecular basis of this heterogeneity. When we immunoprecipitated p30s from labelled cell lysates utilizing goat anti-p30 sera it was observed that (i) in contrast to the four virion p30s, there were only three intracellular p30s (pI 6·0, 6·3 and 6·6), (ii) there was a threefold greater amount of the intracellular compared to the virion form of p30 with pI 6·0, (iii) tryptic peptide maps of both virion and intracellular p30s labelled either with [35S]methionine or 125I showed basically similar patterns with only slight differences in intensity among certain peptides for the p30s with pI 6·1, 6·3 and 6·6, and (iv) the intracellular p30 with pI 6·0 had a peptide that was not present in any of the other p30s. These results suggest that due to some as yet uncharacterized modification(s) of p30 and/or some structural differences between different p30s, a heterogeneity in pi exists. This may be important for assembly of the virion capsid. However, it is also possible that the p30 heterogeneity reflects the presence of multiple M-MLV proviruses within each of the infected cell clones.
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Expression of a Provirus of Human T Cell leukaemia Virus Type I by DNA Transfection
SUMMARYWe isolated the full length provirus of human T cell leukaemia virus type I (HTLV-I) from MT-2, a lymphoid cell line producing HTLV-I. In three non-lymphoid cell lines (COS7, human osteosarcoma HOS cells, and HeLa) this provirus expressed a transacting activity after co-transfection with a recombinant plasmid carrying a bacterial chloramphenicol acetyltransferase gene under the control of a long terminal repeat of HTLV-I provirus. The trans-acting protein p40 was detected by immunoprecipitation in transfected HOS cells. Structural proteins of HTLV-I, the gag and env products, were also formed and processed in the same manner as observed in MT-2 cells. In transfected HeLa cells, the p40 protein was mainly localized in the nucleus, while other structural proteins were detected in the cytoplasm and/or the membrane by indirect immunofluorescence. Syncytium formation was observed in HeLa cells after transfection. These results demonstrated that non-lymphoid cells could produce the major proteins of HTLV-I after DNA transfection of the cloned provirus.
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Suppression of in vitro Neutrophil Function by Feline Leukaemia Virus (FeLV) and Purified FeLV-p15E
More LessSUMMARYFeline neutrophils (PMN) were isolated and exposed to ultraviolet light-inactivated feline leukaemia virus (UV-FeLV) and purified envelope component pl5E (FeLV- pl5E). Functional capacity of exposed PMN was measured in vitro utilizing the chemiluminescence (CL) response. PMN exposed to UV-FeLV demonstrated depressed CL responses to Ca2+-ionophore A23187 and latex particles. However, FeLV-pl5E produced significant suppression in the CL response to A23187 but failed to produce significant alterations in response to latex particles. The data indicate that FeLV-pl5E may, in part, be responsible for increased morbidity and mortality among FeLV-infected cats through suppression of the PMN population.
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Synthetic Vaccines against Friend Murine Leukaemia Virus-induced Erythroleukaemia: in vivo and in vitro Studies with Synthetic Oligopeptides and Sequence-specific Antisera
More LessSUMMARYBiological activities of antisera against synthetic oligopeptides were examined. The peptide antisera were directed against amino acids 6 to 12 (pep1), 124 to 131 (pep2), 256 to 262 (pep3), 283 to 290 (pep4) and 434 to 441 (pep5) of the viral envelope glycoprotein (gp70). Peptide-specific antisera did not neutralize viral infectivity. However, antibodies to pep4 and pep5, which bound to the hydrophobic part of gp70, mediated the complement-dependent lysis of Friend murine leukaemia virus (FLV)-infected cells. STU mice were immunized against FLV-induced erythroleukaemia with synthetic oligopeptides. Vaccines containing only one of these peptides (single-peptide vaccines) as well as multi-peptide vaccines containing pep1, −4, −5 or pep1, −2, −3, −4, −5 were used. Whereas the immunizations with single-peptide vaccines did not protect the immunized mice from FLV-induced erythroleukaemia, multi-peptide vaccines enhanced the survival rate and incubation period after FLV challenge. These results revealed that immunological reactions distinct from neutralizing antibodies can be evoked by immunization with synthetic peptides and can confer limited protection against FLV-induced erythroleukaemia.
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Construction and Characterization of Deletion Mutants of Pseudorabies Virus: a New Generation of ‘Live’ Vaccines
More LessSUMMARYVarious deletions were introduced into a cloned subgenomic fragment (BamHI-1), located in the unique short (Us) region of the DNA from the virulent Northern Ireland Aujeszky-3 (NIA-3) strain of pseudorabies virus (PRV). In the cloned Hin dIII-B fragment, the MluI -BglII fragment was replaced by different MluI-BglII fragments of the deleted BamHI-7clones. Transfection of the deleted HindIII-B fragments together with the HindIII-A fragment of either the NIA-3 or the non-virulent NIA-4 strain yielded replication-competent deletion mutants. The region in Us in which sequences were deleted specified several mRNAs. Some of the mRNAs present in cells infected with NIA-3 were absent from cells infected with the deletion mutants, whereas other differently sized mRNAs were generated. The mutants were examined with respect to their biological properties in cell culture, mice and pigs. The results showed that (i) the type of cytopathic effect induced in cell culture seemed to be determined by the ULregion, (ii) using the mean time to death in mice as a parameter, markers for virulence were present in the Us and UL regions and (ii) the introduction of deletions in Usstrongly reduced the virulence of PRV for pigs. Despite the impaired capacity of the deletion mutants to induce high titres of neutralizing antibodies in the serum, inoculation with mutants derived from NIA-3 prevented clinical disease in pigs upon challenge with the virulent parent strain. These deletion mutants provide a good basis for the production of bioengineered live PRV vaccines.
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Epitope Mapping of the Major Epstein-Barr Virus Outer Envelope Glycoprotein gp350/220
More LessSUMMARYTo understand the complete immunochemical structure of the Epstein-Barr virus (EBV) major membrane glycoprotein gp350/220, monoclonal antibodies (MAbs) reacting with this important viral antigen were isolated. Through competitive inhibition binding studies, it was determined that a group of 18 IgG MAbs recognized seven distinct epitopes on the gp350/220 molecule. Eight of these MAbs fell into a single epitope group with four of those MAbs, as well as a single MAb from another epitope group, being capable of neutralizing EBV strain B95–8 transformation of umbilical cord lymphocytes.
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Demonstration and Mapping of Highly Carbohydrate-dependent Epitopes in the Herpes Simplex Virus Type 1-Specified Glycoprotein C
SUMMARYThe carbohydrate dependence of epitopes in the herpes simplex virus type 1-specified glycoprotein C (gC) was studied using a new solid-phase assay procedure. Glycoprotein C, coated on 96-well microtitre plates, was treated with sialidase and increasing concentrations of periodate. A sequential removal of peripheral monosaccharides from the oligosaccharides of gC was ascertained by an enzyme-linked lectin assay. By using a panel of gC-specific monoclonal antibodies in ELISA, it was found that gC contained two types of epitopes differing in their dependence on terminal galactose and sialic acid for expression. Control experiments indicated that the carbohydrate-dependent epitopes were peptide structures and that the carbohydrates did not directly participate in the antibody-binding reaction. The carbohydrate-dependent epitopes were mapped to antigenic site II, according to the proposed nomenclature, whereas those expressed also in the absence of peripheral sugars were located mainly in antigenic site I. These results were compatible with the relative distribution of oligosaccharides in the gC molecule.
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Restricted Replication of Herpes Simplex Virus Type 1 in Murine Embryonal Carcinoma Cells
More LessSUMMARYHerpes simplex virus type 1 (HSV-1) has a broad host range but the KOS strain of HSV-1 did not replicate efficiently in murine embryonal carcinoma (EC) cells. The yield of infectious HSV-1 from EC cells was 100- to 1000-fold lower than that from fibroblast cell lines of mouse, monkey or human origin. The thymidine kinase (TK) gene of HSV-1 is expressed early during the infectious cycle. The levels of TK mRNA and of TK activity in infected EC cells were only two- to threefold lower than levels from infected fibroblast cells. Infected EC cells supported replication of about half as much HSV-1 DNA as did fibroblast cells. The reduced yield of infectious virus was consistent with a paucity of virions in infected EC cells examined by electron microscopy, suggesting a major block late during the HSV-1 infectious cycle. We isolated a variant strain of HSV-1, called KOSEC, which replicated as efficiently in EC cells as in mouse fibroblasts. KOSEC infected EC and fibroblast cells, synthesized more TK mRNA, more TK enzyme, and more HSV-1 DNA than did the same cells infected with the KOS stain. Both HSV-1 strains induced similar levels of synthesis of gD, an early viral glycoprotein. By co-infection of EC cells with the KOS and KOSEC virus, both the elevated virus yield and the elevated TK synthesis seen in KOSEC-infected cells appeared to be recessive. Apparently a viral mutation that affects expression of some early viral functions can also overcome the EC cell restriction to HSV-1 replication.
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Macaque Monkey Type D Retrovirus Replicates in vitro in a Distinct Subpopulation of B Lymphocytes
More LessSUMMARYType D retroviruses have recently been shown to induce a wasting syndrome with associated lymphadenopathy, thymic atrophy and transient decreased peripheral blood lymphocyte blastogenic responsiveness in juvenile macaque monkeys. The replication in vitro of D/New England virus was assessed in various lymphocyte subpopulations to determine the possible pathogenesis of the immune dysfunction induced by this virus. While D/New England did not replicate in cultured T lymphocytes or induce any demonstrable dysfunction of T cells in vitro, it did grow in the cells of the B lymphocyte lineage. D/New England growth occurred in vitro in African Burkitt’s lymphoma and pre-B cell lines, but not in Epstein-Barr virus-transformed normal B lymphocytes. The infection of a restricted B lymphocyte population by this primate type D retrovirus may play a role in the aetiology of the immune abnormalities which it induces.
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Ultrastructural Studies of Kunjin Virus-infected Aedes albopictus Cells
More LessSUMMARYUltrastructural changes in Aedes albopictus cells infected with Kunjin virus were characterized from 12 to 72 h post-infection. Early in infection (16 h), there were no prominent ultrastructural changes except for an increase in the number of vacuoles in the cytoplasm. As the infection progressed the rough endoplasmic reticulum appeared to lengthen and whorls of fibres were observed within some vacuoles. Virus particles were observed in small numbers scattered in the cytoplasm between 24 to 30 h after infection. However, by 48 h, large numbers of morphologically mature virus particles were visible, usually in association with the rough endoplasmic reticulum, with the clusters of vesicles or with convoluted smooth membranes. Although no definite evidence of precursor particles or of budding of virions was obtained, the clusters of vesicles and the various membranous structures appeared to play a role in the morphogenesis of this virus. The results are compatible with the hypothesis that maturation of virus particles occurs within all these virus-induced structures.
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Integration and Transcription of Human Papillomavirus Type 16 and 18 Sequences in Cell Lines Derived from Cervical Carcinomas
More LessSUMMARYFive cell lines, SKG-I, SKG-II, SKG-IIIb, QG-U and QG-H derived from cervical carcinomas of Japanese patients, were examined for the presence of human papillomavirus (HPV) DNA and the expression of viral mRNA. The DNA of HPV type 16 was shown to be linked covalently with SKG-IIIb, QG-U and QG-H cell DNA, and HPV 18 DNA with SKG-I and SKG-II cell DNA. Although different regions of the HPV genome were integrated in these cell lines, the non-coding region and an early region including the E6 and E7 open reading frames (ORFs) were conserved in all cell lines. The complete genome of HPV 16 was found in QG-H cells by digestion of the DNA with a single-cut restriction enzyme. The other early region ORFs E1, E2, E4 and E5 were interrupted by flanking host cell DNA, suggesting that the integration into host cell DNA occurs preferentially in this region. HPV-specific mRNA species were detected in all five cell lines. In the three cell lines containing the HPV 16 genome, mRNAs hybridized with the early region of the genome, covering the entire E6 and E7 ORFs and a minor part of the E1 ORF, although the amount and size of the major mRNAs varied in these cell lines. These mRNAs did not hybridize with the late region of the HPV genome containing the L1 and L2 ORFs. In SKG-II, SKG-IIIb and QG-H cells we also detected c-myc and c-Ha-ras mRNA expression at about nine times the level of that in normal cells.
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Role of Prostaglandins and Non-steroid Anti-inflammatory Drugs in the Pathogenicity of Vaccinia Virus
More LessSUMMARYThe effect of prostaglandins (PGs) of the A series (A1 and dimethyl PGA2), E1, D2, F2α and PGI2 (prostacyclin) and of inhibitors of PG synthesis (aspirin and indomethacin) on the pathogenicity of vaccinia virus was studied in BALB/c mice. PGs of the A series, D2 and F2α conferred little or no protection to mice against the lethal effects of vaccinia virus. Mice treated with PGE1 showed a dramatic increase in mortality after viral infection. However, when mice were treated with PGI2, their survival was greatly enhanced. Mice treated with aspirin and indomethacin showed a marked increase in mortality. Increased mortality correlated with higher virus yields in target tissues (spleen) and with inhibition of antibody response, whereas the increase in survival correlated with lower virus yields and with normal antibody responses. The significance of our findings is that PGI2 can block the outcome of the disease caused by vaccinia virus whereas other PGs and their inhibitors not only worsen the disease, but may activate and enhance viral infections through immune suppression.
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Small Circular Single-stranded DNA Associated with Foliar Decay Disease of Coconut Palm in Vanuatu
More LessSUMMARYThe single-stranded DNA previously reported to be associated with foliar decay disease of coconut palm appears to be predominantly circular, on the basis of its behaviour in two-dimensional polyacrylamide gel electrophoresis, electron microscopy, and its resistance to end-labelling following treatment with alkaline phosphatase and polynucleotide kinase. The DNA sedimented at between 12S and 15S and had a contour length for the circular molecules consistent with the predominant DNA having a molecular weight of about 0·43 × 106, and comprising approximately 1300 nucleotides. These properties differ from the genomic DNAs of known plant viruses.
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Wheat Streak Mosaic Virus Cylindrical Inclusion Body Protein
More LessSUMMARYA protein of apparent molecular weight 66000 was purified from wheat plants infected with wheat streak mosaic virus. Antiserum to this protein, labelled with gold, specifically stained cylindrical inclusions in ultrathin sections of virus-infected cells. Antiserum to the M r 66000 protein did not react with capsid protein in Western blots, nor did antiserum to capsid protein react with the M r 66000 protein. Both antisera reacted with homologous antigens. The concentration of the M r 66000 protein in extracts from infected leaves was about 100 μg per g of leaves, which is higher than the usual concentration of virions.
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Replication of Red Clover Necrotic Mosaic Virus RNA in Cowpea Protoplasts: RNA 1 Replicates Independently of RNA 2
More LessSUMMARYInoculation of cowpea mesophyll protoplasts with unseparated RNA 1 and RNA 2 from red clover necrotic mosaic virus in the presence of polyethylene glycol resulted in virus RNA replication, the synthesis of virus capsid polypeptide and the formation of virus particles; 75 to 85% of the viable protoplasts became infected and the yield of virus particles or virus RNA after 72 h incubation corresponded to about 3 × 106 virus genomes per infected protoplast. In contrast, no replication could be detected when protoplasts were inoculated with RNA 2 alone. However, inoculation of protoplasts with RNA 1 alone resulted in its replication and the formation of virus particles, with a yield similar to that obtained after inoculation with both RNAs. Since infection of plants requires both RNA 1 and RNA 2 to be present, the demonstration of the independent replication of RNA 1 in single cells strengthens the hypothesis that RNA 2 plays a role in the cell-to-cell transmission of the virus.
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Nucleotide Sequence of the Capsid and Nuclear Inclusion Protein Genes from the Johnson Grass Strain of Sugarcane Mosaic Virus RNA
More LessSUMMARYThe nucleotide sequence of the 3′ terminal 1782 nucleotides of sugarcane mosaic virus (SCMV) genome has been determined. There is an open reading frame, from the 5′ end, of 1307 nucleotides upstream from a 475 nucleotide 3′ non-coding region that is polyadenylated. The open reading frame encodes a polypeptide of 435 amino acids.The segment of the genome encoding the viral capsid protein (mol. wt. 34200) is adjacent to the 3′ non-coding region. The predicted capsid protein is similar in sequence to the capsid protein sequence predicted for tobacco etch virus (TEV). Part of another protein encoded in the same reading frame, similar to the predicted nuclear inclusion protein from TEV, has been identified upstream from the coat protein gene.The results indicate that the genome of SCMV encodes one or more large proteins that are processed to the mature proteins.
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