- Volume 64, Issue 3, 1983
Volume 64, Issue 3, 1983
- Animal
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Prostaglandins Enhance Intercellular Adhesion of Vero Cells Infected with Herpes Simplex Virus
More LessSUMMARYThe effect of prostaglandins (PGs) and cyclic nucleotides on adherence between uninfected Vero cells or between uninfected cells and cells infected with wild-type or syncytial strains of herpes simplex virus (HSV) was studied. In the absence of serum, PGE2 increased adhesion between uninfected cells whereas PGF2α decreased it. However, both PGE2 and PGF2α increased adhesion between cells infected with HSV and uninfected cells. Moreover, dibutyryl cyclic AMP increased adhesion between uninfected cells and cells infected with HSV, whereas dibutyryl cyclic GMP decreased it.
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Ribonucleotide Reductase Induced by Herpes Simplex Virus has a Virus-specified Constituent
More LessSUMMARYRibonucleotide reductase, an enzyme found in all prokaryotic and eukaryotic cells that synthesize DNA, is induced by herpes simplex virus (HSV). In this study the effect of anti-HSV antiserum on the induced ribonucleotide reductase has been examined and the ability of different temperature-sensitive (ts) mutants of HSV-1 to induce the enzyme has been investigated. The HSV-1-induced ribonucleotide reductase was inhibited by antiserum raised against infected cell lysates but not by preimmune serum. The wild-type (ts +) virus induced similar levels of ribonucleotide reductase at 31 °C and 38.5 °C (the permissive and non-permissive temperatures respectively for the ts mutants). All ts mutants induced approximately wild-type levels of the enzyme at 31 °C. At 38.5 °C, two of the four ts mutants studied also induced wild-type levels of enzyme but ts G failed to induce any activity while ts K induced variable but low levels. The enzyme activity induced by ts G at 31 °C was thermolabile both in vivo and in vitro. These results provide the first strong evidence that the induced ribonucleotide reductase activity is at least partially virus-coded.
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Characterization of Abnormal Thymidine Kinases Induced by Drug-resistant Strains of Herpes Simplex Virus Type 1
More LessSUMMARYTwo TK+ acyclovir-resistant variants of herpes simplex virus (HSV) (S1 and Tr7) and one TK+ BVdU-resistant variant (B3) induce abnormal thymidine kinases with impaired ability to phosphorylate the drugs used in their isolation. These enzymes have been purified and their properties compared with those of the wild-type (wt) parent, SC16. The enzyme induced by S1 differed markedly from the other three in both its responses to salt and to pH. B3 TK recognized the enzyme’s natural substrates, thymidine, deoxycytidine, dTMP and ATP as well as the wt enzyme. In contrast, Tr7 and S1 TKs failed to bind deoxycytidine and bind thymidine less well than wt. Tr7 and S1 TKs had affinities for dTMP similar to those of B3 and the wt enzymes. ATP binding to wt, Tr7 and B3 enzymes was similar but this substrate bound only weakly to S1 TK. Each mutant displayed a characteristically distinct pattern of affinities for a range of nucleoside analogue substrates, suggesting that they will show some cross-resistance to drugs which have a similar mechanism of action to acyclovir and BVdU.
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Cell-bound and Circulating IgE Antibody to Herpes Simplex Virus
More LessSUMMARYMice immunized with ultraviolet-inactivated herpes simplex virus (HSV) or injected with infectious virus developed IgE-specific antibody to HSV. Cell-bound IgE was detected by measuring the release of histamine from peritoneal mast cells challenged in vitro with virus antigens. Circulating IgE antibody was detected by sensitizing rat basophilic leukaemia (RBL-2H3) cells with sera from HSV-immunized mice and then challenging these cells with virus or control antigens to release histamine. IgE antibody may contribute to the pathogenesis of virus infections.
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Can Epstein–Barr Virus Infect and Transform All the B-Lymphocytes of Human Cord Blood?
M. Zerbini and I. ErnbergSUMMARYQuantitative aspects of Epstein—Barr virus infection and transformation of human neonatal B-lymphocytes have been investigated. 72 to 90% B-cells were obtained with enrichment. Of the B-cells, 19 to 97% showed nuclear antigen (EBNA) 2 days after infection. A difference between different B-cell donors in susceptibility to infection was noted. Analysis of the virus dose—response curves obtained with twofold virus dilutions showed that one virus particle is sufficient to induce EBNA in a cell. Of the infected cells, 50 to 95% multiplied in microtitre wells containing a human fibroblast feeder layer, while only a small proportion established growing colonies in soft agarose, that could be picked up and subcultured.
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Molecularly Cloned Bovine Papillomavirus DNA Transforms Mouse Fibroblasts in Vitro
More LessSUMMARYNIH 3T3 mouse fibroblasts were transformed in vitro by transfection with viral DNA from bovine papillomavirus (BPV) types 4, 2 and 1. The viral DNAs were molecularly cloned in pAT153 to construct pBV4, a recombinant plasmid containing the whole genome of BPV4, pBV2 containing the entire genome of BPV2, and pBV1-D1 which contains the 69% transforming DNA fragments of BPV1. The transformed cells lost contact inhibition, were anchorage-independent, required low serum and were tumourigenic in nude mice. This is the first report of cell transformation in vitro by BPV4 DNA. The recombinant plasmids transformed NIH 3T3 cells with an efficiency of 200 to 300 foci/µg DNA. Cleavage of the recombinant plasmids with enzymes that separate the viral DNA from pAT153 DNA did not significantly alter the efficiency of transformation. In all the transformed cells analysed, the recombinant plasmids were found as multiple copies of non-integrated monomers.
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Virosomes Constructed from Lipid and Purified Friend Leukaemia Virus Glycoprotein
More LessSUMMARYLiposomes were loaded by a dialysis technique with purified envelope glycopolypeptide, gp85, of the Friend murine leukaemia virus (F-MuLV). The gp85 liposomes prepared from cellular lipid had a buoyant density of 1.05 g/ml and an apparent diameter of 50 to 300 nm. The gp85 was not simply entrapped by liposomes nor adsorbed non-specifically to their outer surface. Experiments with radioactively labelled protein, electron microscopic examinations, protease treatment and concanavalin A binding showed that gp85 is anchored in the liposomal membrane and orientated asymmetrically as in the virus envelope. Moreover, gp85-covered liposomes displayed some functions of the intact F-MuLV envelope, such as absorption of antibodies to gp70 and haemagglutination after enzyme treatment. To some extent, these lipid vesicles appeared to be reconstituted F-MuLV envelopes and thus, by analogy to other systems, were named retrovirosomes.
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A Deletion Mutant of Mouse Mammary Tumour Virus, Lacking 516 Nucleotides of the 5′ Long Terminal Repeat Sequence, Can Be Expressed in a Hormone-responsive Fashion
More LessSUMMARYIn vitro manipulation of proviral DNA of mouse mammary tumour virus (MMTV) was used to construct mutants with defined deletions at the 5′ end of the proviral gene. In the mutants 516, 1400 and 2000 nucleotides were removed from the 5′ end. The deleted proviral DNA was tested for transcription and glucorticoid hormone regulation of viral RNA expression upon cotransfection into rat XC tk− cells with a thymidine kinase gene. Intact proviral DNA contained in the plasmid vector pBR322 and the deletion mutant pGR16Δ516, missing 516 nucleotides of the 5′ long terminal repeat (LTR) sequence, were transcribed in a hormone responsive fashion and produced RNA species of 35S and 24S. Deletion of the entire LTR sequence abolished MMTV transcription and the hormonal effect.
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Analyses of Structural Polypeptides of Seven Different Isolates of Influenza C Virus
More LessSUMMARYThe major structural polypeptides (gp88, NP and M) of seven different influenza C virus strains isolated between 1947 and 1981 in U.S.A. and Japan were compared by SDS-polyacrylamide gel electrophoresis and one-dimensional mapping of the peptide fragments produced after limited proteolysis with various proteases. Of the three polypeptides analysed, the membrane (M) protein appeared to be the most highly conserved since the electrophoretic mobility as well as the mapping pattern of this protein was found to be identical among all seven strains. The structure of nucleoprotein (NP) was also found to be highly conserved. The proteins of five isolates from 1964 to 1981 showed migration rates and mapping patterns indistinguishable from each other though they were slightly different in mapping patterns from the earlier isolates, C/Taylor/1233/47 and C/JJ/50. The similarities between influenza C strains were also evident with the surface glycoprotein, gp88. The gp88 proteins of the five strains isolated in 1947, 1950, 1971 and 1981 were virtually identical in migration rates as well as in mapping patterns, while the two isolates of 1964 and 1974 showed minor differences. These results strongly suggest that the surface glycoprotein of influenza C virus is structurally much more stable than the haemagglutinin and neuraminidase glycoproteins of influenza A and B viruses. Further, the findings that differences from the original influenza C strain, Taylor/1233/47 were detectable in the strains isolated in 1964 and 1974 but not in the strains isolated in 1971 and 1981 suggest that unlike the antigenic drift of types A and B influenza viruses, the structural variation of gp88 may not be a sequential event.
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Distribution of Viral Antigen within the Lower Respiratory Tract of Ferrets Infected with a Virulent Influenza Virus: Production and Release of Virus from Corresponding Organ Cultures
More LessSUMMARYUsing fluorescent antibody techniques, a semi-quantitative survey has been made of the distribution of influenza virus antigen in the trachea, main bronchi, and three zones (hilar, intermediate and alveolar) of all four lung lobes of ferrets following intranasal inoculation of a virulent clone (7a) of the recombinant influenza virus A/PR/8/34-A/England/939/69 (H3N2). The results confirm the indications from our previous quantitative surveys of infectious virus and histological damage in these areas, namely that infection is confined largely to airway epithelium and is rare in the alveoli. Furthermore, in the lung zones, viral antigen resided mainly in the bronchial rather than bronchiolar epithelium. In attempts to identify the reasons for lack of alveolar involvement organ cultures of alveolar tissue, from which all major airways had been removed, produced levels of virus similar to cultures of bronchus and trachea and the hilar and intermediate lung zones which contain airway and alveolar tissue. Hence, the lack of alveolar infection in vivo must be due to factors which prevent virus attack of susceptible alveolar cells. However, these organ culture experiments showed that a contributing factor could be very poor release of virus from any alveolar cells that do become infected. In contrast, although cultures of bronchi produced less virus than those of nasal turbinates (the most susceptible tissue in vivo) they released a high proportion of their yield and this ease of release may contribute to spread of infection in vivo.
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Lymphocytic Choriomeningitis Virus. III. Structural Proteins of the Virion
More LessSUMMARYAnalysis of radioactively labelled and highly purified infectious lymphocytic choriomeningitis (LCM) virus by polyacrylamide gel electrophoresis (PAGE) revealed 12 components which, according to their apparent molecular weight and glycosylation status, were designated as p19, p25, p26, gp35, p38, gp44, gp60, p63, p77, gp85, gp130, and p200. As shown by immunoprecipitation, they all bound to rabbit anti-LCM virus antibodies. Three proteins, namely gp35 (= ‘GP-2’), gp44 (= ‘GP-1’) and p63 (= ‘NP’), had previously been described by others as major constituents of the virion. Our results confirm this and suggest that gp60, p77, gp85, and p200 are further distinct structural proteins. In contrast, p25 and p38 appear to be cleavage or degradation products of p63; p19 and p26 seem to belong to gp60, which could be the monomeric form of a dimer, gp130. Peptide mapping by limited proteolysis revealed considerable overlapping of amino acid sequences among the major glycoproteins with one peptide being common to all. From the results of PAGE performed after external labelling of intact virions, we conclude that gp44, gp60, and gp85 (but not gp35) form the surface of the virus envelope. Analytical isoelectric focusing under non-reducing conditions has shown that the major glycoproteins appeared to consist of several components with different isoelectric points.
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Unusual Neutral Oligosaccharides in Mature Sindbis Virus Glycoproteins are Synthesized from Truncated Precursor Oligosaccharides in Chinese Hamster Ovary Cells
More LessSUMMARYWe have previously demonstrated the presence of unusual small asparaginyloligosaccharides [(Man)3GlcNAc2-ASN] in the mature glycoproteins of Sindbis virus released from both wild-type and lectin-resistant Chinese hamster ovary cells, but the mechanism of synthesis of these structures was not determined. Gel filtration and endo-β-N-acetylglucosaminidase analyses of Pronase-digested glycopeptides from [3H]mannose-labelled Sindbis virus released at different times after infection of a phytohaemagglutinin-resistant line of Chinese hamster ovary cells demonstrated that these small asparaginyl-oligosaccharides were present in similar relative amounts in virus released throughout the virus infection, rather than arising primarily at late times when cytopathic effects were maximal. Similar analyses of pulse-labelled, cell-associated viral glycopeptides suggested that these small oligosaccharides on mature virus glycoprotein resulted from the normal α1,2-mannosidase processing of truncated precursor oligosaccharides (containing five rather than nine mannoses), rather than from aberrant processing or degradation of the full-size precursor oligosaccharides or normal intermediates.
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Ontogeny of Yellow Fever 17D Vaccine: RNA Oligonucleotide Fingerprint and Monoclonal Antibody Analyses of Vaccines Produced World-wide
More LessSUMMARYYellow fever 17D vaccines are currently manufactured with approval of the World Health Organization (WHO) in 11 countries. These vaccines have proven highly efficacious and safe. Nevertheless, they have not been fully characterized genetically, a problem for future standardization and modernization of vaccine manufacture now being proposed by WHO. Vaccines in use are derived from two distinct substrains (17D-204 and 17DD) which represent independently maintained passage series from original 17D. In this study, all 17D vaccines produced world-wide were characterized by RNA oligonucleotide fingerprinting. Forty-two large oligonucleotides were compared, and differences from an arbitrarily selected reference strain (produced by Connaught Laboratories in the U.S.A.) were determined. With one exception (vaccine produced in South Africa), fingerprints of vaccines derived from substrain 17D-204 were identical. The South African primary seed differed in position of one oligonucleotide, reflecting a charge shift due to a single base change. This difference occurred within one egg passage; a further change in the South African vaccine occurred within one or two passages from primary seed. No antigenic differences between 17D-204-derived vaccines (including South Africa) were demonstrated by neutralization tests using monoclonal antibody. Vaccines derived from the 17DD substrain consistently differed from 17D-204 vaccines in the absence of one oligonucleotide (No. 37). This change probably occurred during 40 additional egg passages in development of the 17DD vaccines. A clear antigenic difference was shown between 17D-204 and 17DD substrain vaccines using monoclonal antibody. 17DD vaccines showed minor genotypic differences, suggesting a higher degree of genetic instability than 17D-204 vaccines. No oligonucleotide fingerprint differences were found between avian leukosis virus (ALV)-free and ALV-contaminated vaccines. No definite genomic correlate of neurovirulence was defined by fingerprinting strains with a history of encephalitic complications in man or of failure to pass monkey neurovirulence tests. Parent Asibi virus showed several oligonucleotide differences and was serologically distinct from 17D vaccine.
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Rapid Baculovirus Detection, Identification, and Serological Classification by Western Blotting-ELISA using a Monoclonal Antibody
More LessSUMMARYA hybridoma cell line, called 3D10, has been established which secretes monoclonal antibodies against the 42K protein of Autographa californica nuclear polyhedrosis virus (Ac NPV). Following separation of a complex protein mixture by denaturating SDS-polyacrylamide gel electrophoresis and electrophoretic protein transfer to a nitrocellulose filter, this monoclonal antibody is still able to react with its antigen. The antigen-antibody complex can be stained by indirect enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-conjugated rabbit anti-mouse serum. The Western blot—ELISA described is extremely sensitive, being able to detect as little as 10 ng of Ac NPV or 3 × 105 polyhedral inclusion bodies. Using this method it is possible to find traces of Ac NPV in very crude materials, e.g. impure polyhedra, infectious haemolymph, and larval homogenates. On the other hand, this monoclonal antibody is very specific and reacts only with isolates of Ac NPV and Galleria mellonella NPV. No reactions were found against eleven other NPV isolates. The implications of these findings for baculovirus identification and classification are discussed.
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Molecular Basis of Rabies Virus Virulence. II. Identification of a Site on the CVS Glycoprotein Associated with Virulence
More LessSUMMARYNine anti-G monoclonal antibodies were used to select mutants of the CVS strain of rabies virus resistant to neutralization. Seven mutants were avirulent in adult mice and two others exhibited an attenuated pathogenicity. Both categories were resistant to monoclonal antibodies 194-2 and 248-8. Virulence appears to be associated with a particular configuration of a region of the glycoprotein which is located at the intersection of the epitopes recognized by these two monoclonal antibodies. Our results confirm the role played by the glycoprotein in the neurovirulence of rabies virus.
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Production of Human Monoclonal Antibody to X31 Influenza Virus Nucleoprotein
SUMMARYIn vitro stimulation of human peripheral blood mononuclear cells with X31 influenza virus antigen has been used to enrich for specific anti-X31 antibody-producing cells. Following Epstein—Barr virus transformation of these stimulated cells, a cell line which produces human antibody to X31 virus was derived and subsequently cloned. The cloned cells secrete an IgG1 κ antibody which is directed against the nucleoprotein of A type influenza virus. Culture supernatants contain 10 to 20 µg/ml of specific antibody which is now used as a standard for the ELISA assay used in our laboratory to detect antibodies to influenza virus.
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Analysis of Theiler’s Virus Isolates from Persistently Infected Mouse Nervous Tissue
More LessSUMMARYThe DA strain of Theiler’s virus causes a chronic progressive demyelination in mice following intracerebral inoculation. Virus was isolated from chronically infected mice, and then grown in cell culture, and the isolates were compared with the parent virus used for inoculation. No defective interfering particles or temperature-sensitive virus were recovered, and capsid proteins appeared identical by SDS–PAGE. One of three isolates had evidence of genomic mutation by T1 ribonuclease oligonucleotide fingerprinting. The significance of these findings with regard to the generation and maintenance of persistence and to adaptation to cell culture is discussed. Also of interest was the marked difference between the DA fingerprint and that of GD VII, a serologically related strain with different biological activity.
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Interferon Induces the Production of Membrane Protein-deficient and Infectivity-defective Vesicular Stomatitis Virions through Interference in the Virion Assembly Process
More LessSUMMARYThe reduced rate of synthesis, maturation and degradation as well as the level of accumulation of the intracellular virus proteins in VSV-infected cells may account for the overall reduction (less than 10-fold) of progeny virion yield due to interferon (IFN); however, the deficiency of the virions proteins, G and M, which apparently caused a drastic loss of infectivity of these progeny virions (about 1000-fold) cannot be easily explained, because the concentrations of G and M proteins relative to other virus proteins were not reduced in the cell. In fact, intracellular M protein was significantly increased. Moreover, the virus proteins in IFN-treated and control cells were synthesized and accumulated in large excess of the amount incorporated into the released virions. The reduction in the intracellular activity of GlcNac-P-P-Dol transferase did not appear to play a direct role in the antiviral mechanism in this system. Our results, however, do suggest that the deficiency of G and M proteins in the virion is related to specific inhibition of the incorporation of either or both of these proteins in the virus assembly process.
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Pathogenesis of Mouse Scrapie: Evidence for Spread of Infection from Central to Peripheral Nervous System
More LessSUMMARYThe onset of replication has been studied in spinal cord, dorsal root ganglia and spinal nerves of CW mice infected intraperitoneally with the 139A strain of scrapie. The patterns obtained suggest a centrifugal spread of infection from central to peripheral nervous systems. Infectivity titres in the peripheral nervous system reached a plateau long before the end of the incubation period, and the maximum titres were much lower than in the central nervous system. This suggests that there is a restriction of replication in the peripheral nervous system similar to that already known in extraneural tissues.
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Intracellular RNA Expressed in Black Beetle Virus-infected Drosophila Cells
More LessSUMMARYBlack beetle virus replicates in Drosophila cells with the synthesis of virus-specific RNA and protein being detected as early as 2 h post-infection and continuing for at least 48 h. Shut-off of host synthesis did not occur in infected cells until 8 to 10 h post-infection. Examination of the RNA from infected cells on denaturing gels or on agarose/acrylamide gels demonstrated the production of four RNA species not found in uninfected cells. Two of these species, being single-stranded, co-migrated with the virion RNA and the other two were the double-stranded replicative stages of the virion RNA. No additional RNA species were found in infected cells, either in the presence or absence of actinomycin D.
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Volume 105 (2024)
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