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Volume 64,
Issue 11,
1983
Volume 64, Issue 11, 1983
- Articles
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- Animal
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Four Independent Antigenic Determinants on the Capsid Polypeptides of Aphthovirus
More LessSUMMARYFour independent antigenic determinants in the virus capsid have been defined in radioimmunoassay tests using antisera prepared against isolated structural polypeptides VP1, 2 and 3. Only one determinant was detected on the intact particle and it was contained within VP1 (called the VP1-A determinant). Three determinants were found on the 12S subparticle, two contained within VP1 (VP1-A and VP1-B determinants) and one contained within VP2 (VP2 determinant). The fourth determinant was contained within VP3 (VP3 determinant) and could only be detected when the virus was disrupted to individual polypeptides. It is concluded that the VP1-A determinant is likely to be of epidemiological importance since (i) it was present on the intact virion, (ii) variation at this determinant was detected with heterologous field strains, (iii) it contained a determinant responsible for the induction of neutralizing antibodies and (iv) it was an antigenic component of the trypsin-sensitive region of VP1 which has been shown previously by several workers to be of critical importance in the immunogenicity of the virus.
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Location of an Immunizing Determinant within Polypeptide VP1 of Type O Aphthovirus
More LessSUMMARYVP1 is the only structural polypeptide of aphthovirus able to stimulate the production of neutralizing antibody. The region of VP1 responsible for this activity was located by testing various proteolytic fragments of VP1 for their ability to compete for virus-specific antibodies in serum raised against the intact polypeptide. No antigenic activity could be detected in VP1 fragments isolated from trypsin-treated virus. Controlled digestion revealed that trypsin cleaved VP1 in four places in a preferred order, whereas chymotrypsin cut at a maximum of two sites. The initial cuts by the two proteases were made very close to each other, and in each case resulted in a greatly reduced affinity of the VP1 fragments for virus-specific antibodies in anti-VP1 serum. In contrast, aphthovirus was resistant to Staphylococcus aureus V8 protease, and treatment of isolated VP1 with this protease generated a peptide of mol. wt. 8500 which competed efficiently with virus for antiserum to VP1 and for an absorbed antiviral serum specific for trypsin-sensitive sites. The results indicate that these antisera interact specifically with aphthovirus at a single antigenic determinant located on VP1 approximately two-thirds of the way along the polypeptide sequence.
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Antigenic and Biochemical Analysis of Field Isolates of Influenza B Virus: Evidence for Intra- and Inter-epidemic Variation
More LessSUMMARYDetailed antigenic analysis using a panel of monoclonal antibodies was carried out on the haemagglutinin antigen of 53 influenza B viruses isolated from an epidemic in a single school. Thirteen distinguishable antigenic groupings of influenza B viruses could be detected but 26 of the viruses were in two groups (III and IV) which co-existed during the entire epidemic. Antigenically distinguishable influenza B viruses were isolated from an epidemic in a second nearby school. Influenza B viruses isolated from the two schools could be further distinguished by different electrophoretic mobilities of NS1 polypeptides and of genes 1, 2, 3 and 6, whereas viruses from a single school epidemic were very closely related as regards these biochemical characteristics. The findings are consistent with the hypothesis that the outbreak was initiated by a single individual who excreted antigenic mutants of which predominantly two spread and coexisted during the epidemic, although the additional occurrence of random mutations during the evolution of the epidemic cannot be excluded.
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Comparison of Cytoskeletal Organization in Canine Distemper Virus-infected and Uninfected Cells
More LessSUMMARYThe organization of vimentin filaments, keratin filaments, microtubules and microfilaments was compared in canine distemper virus (CDV)-infected and uninfected cells by indirect immunofluorescence. Infection of tissue culture cells with CDV caused a total reorganization of all the cytoskeletal structures with the most notable changes in the microtubules and intermediate filaments. During virus infection two different patterns of staining were observed for both the intermediate filaments and microtubules, suggesting a step-by-step reorganization of the structures. While the two types of intermediate filaments (vimentin and keratin) had quite different staining patterns, the vimentin (but not keratin) filaments had a distribution pattern similar to the microtubules in both infected and uninfected cells. These results suggest that microtubules and vimentin (but not keratin) filaments may have a close association in CDV-infected cells.
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Evidence that Avian Tumour Virus-immune Chicken Sera Recognize Only Viral Structural Antigens on the Surface of Avian Tumour Virus-infected Cells
More LessSUMMARYThe specificity of the humoral response of chickens to avian tumour viruses (ATV) was investigated by reacting ATV-immune sera with Triton X-100 extracts of uninfected, infected and transformed chicken embryo fibroblasts. Analysis of these immune reactions by polyacrylamide gel electrophoresis revealed that avian leukosis virus-challenged and Rous sarcoma virus-challenged chickens recognized only two major cell surface antigens of 100000 and 29000 mol. wt. which were present on transformed and non-transformed virus-producing cells. No labelled antigens were precipitated from uninfected cells or transformed cells producing the envelope-defective mutant RSV(-). The antigens were shown to be related to the major envelope glycoproteins of the virus and to contain group-specific determinants common to ATV subgroups B and C. No group-specific determinants common to ATV subgroups A and B or subgroups A and C were detected. Chickens were found to have a strong antibody response to the 100000 and 29000 mol. wt. proteins prior to and during tumour rejection, even in the absence of neutralizing antibody to the challenge virus. No tumour-specific surface antigen distinct from the virion structural antigens was detected by any of the immune chicken sera on any of the transformed cells tested.
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Effect of Dengue Virus Infection on Fc-receptor Functions of Mouse Macrophages
More LessSUMMARYFc-receptor-mediated attachment and ingestion of opsonized sheep erythrocytes (EA) by the macrophages of spleen and peritoneal cavity were studied during dengue virus type 2 (DV) infection of Swiss albino mice. Following intracerebral inoculation, virus antigen could be demonstrated by immunofluorescence in the splenic macrophages from day 4 and in peritoneal macrophages from day 5 post-infection, with a higher number of positive cells discernible on the 7th and 8th days. The virus could be isolated from spleen tissue from day 5. The total number of cells was markedly reduced from day 4 onwards both in the spleen and peritoneal cavity. A loss in the capacity to attach and ingest EA was noticed, the lowest values of attachment index (AI) and phagocytic index (PI) being reached on day 4. At later periods the AI values increased markedly but continued to be significantly less than those in uninfected control mice. The PI values continued to be lower throughout. The dichotomy between the Fc-mediated attachment and ingestion may be a mechanism for prevention of virus infection of macrophages.
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Cell Surface Receptor-mediated Internalization of Interferon: Its Relation to the Antiviral Activity of Interferon
More LessSUMMARYThe binding of [3H]leucine-labelled pure human interferon α from Namalwa cells to human FL and Daudi cells was studied, and evidence obtained to indicate receptor-mediated internalization of interferon. Cell surface-bound and internalized interferons were quantified separately as trypsin-released and unreleased radioactivities, respectively. At 37 °C, surface-bound interferon reached a maximum after 1 h and then decreased, while internalized interferon reached a maximum after 2 h. At 21 °C, in contrast, surface-bound interferon reached a maximum after 2 h and did not decrease thereafter; no internalization was observed. The same was true at 37 °C in the presence of NaF, indicating dependence of internalization on temperature and energy. In control cultures at 37 °C, internalized interferon, after reaching a maximum, decreased after prolonged incubation, and concomitantly acid-soluble radioactivity appeared in the culture medium. The decrease in internalized interferon and the emergence of degraded interferon were inhibited by the lysosomotropic agents, ammonium chloride and chloroquine. The fate of labelled interferon, bound to the cell surface of FL cells at 21 °C, was studied at 37 °C, and the results indicated that trypsin-unreleased interferon was derived from the surface-bound interferon, and was secreted in part into the culture fluid in a degraded form upon prolonged incubation. The relation of internalization of interferon to its biological activity was studied in three ways. In FL cells, the antiviral activity was not induced when internalization of interferon was entirely blocked (at 21 °C or in the presence of NaF at 37 °C). In Daudi cells, both 2′-5′-oligoadenylate (2–5A) synthetase induction by interferon and internalization of interferon were inhibited completely by diethyldithiocarbamate (DDC), whereas in the case of FL cells, DDC inhibited neither 2–5A synthetase induction nor internalization of interferon. Raji cells, which have an interferon-specific binding site on the cell surface but are insensitive to interferon, were found not to internalize interferon, whereas other Burkitt’s lymphoma cells, Daudi and Namalwa, which are sensitive to interferon, did internalize it. These findings suggest (but do not prove) that internalization is required for the establishment of interferon activity.
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Interferon Induction by Viruses. X. A Model for Interferon Induction by Newcastle Disease Virus
More LessSUMMARYUnirradiated Newcastle disease virus (NDV, strain AV) induced high levels of interferon (IFN) in primary chick embryo cells if the cells were ‘aged’ in vitro for 6 to 7 days. Dose (multiplicity)-response (IFN yield) curves, carried out in the presence of anti-NDV serum to prevent cycling infection, revealed that stocks of NDV-AV contain about sevenfold more IFN-inducing particles (IFP) than infectious particles (PFP). These non-infectious IFP were responsible for nearly all IFN induction in ‘aged’ cells, since PFP were determined to be incapable of inducing IFN. In contrast, with mouse L(Y) cells as hosts, about one-third the number of particles as there are PFP appeared to score as IFP. Heat and u.v. radiation (254 nm) inactivated NDV IFP and PFP activity at the same rate whether tested in chick or mouse cells, implying that virion-associated transcription is required to induce IFN. A model is proposed to account for the generation of IFN-inducing particles from infectious NDV following u.v. irradiation, and their subsequent inactivation at high doses of radiation. The model defines a series of u.v. targets in the NDV genome that regulate the expression of IFN-inducing particle activity in ‘unaged’ chick embryo cells.
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Antigen-specific Suppression of Delayed Type Hypersensitivity to Murine Cytomegalovirus in MCMV-infected Mice
More LessSUMMARYWhen primary infection with murine cytomegalovirus (MCMV) was by the intravenous route, the resulting delayed type hypersensitivity (DTH) to MCMV was reduced compared with subcutaneous or intraperitoneal infection. Intravenous injection of 103 or more p.f.u. of MCMV also reduced the DTH response to virus injected at the same time subcutaneously. When spleen cells from mice injected intravenously with MCMV 1 to 3 weeks earlier were transferred to subcutaneously infected mice, the resulting DTH response was suppressed. The cells responsible were T cells, being non-adherent and Thy-1 positive; they were not destroyed by complement plus antibody to either Lyt-1 or Lyt-2. Suppression was not transferred by serum. The suppression was antigen-specific; DTH responses induced by sheep red blood cells and by a thymidine kinase-negative strain of herpes simplex virus type 1 were not significantly suppressed by concurrent intravenous infection with MCMV. Suppression of DTH to MCMV caused by intravenously injected MCMV was reduced in mice given 50 mg/kg cyclophosphamide, but not in those given 200 mg/kg. Deoxyguanosine had no effect on the induction of DTH to MCMV.
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Recurrence Phenotypes and Establishment of Latency Following Rabbit Keratitis Produced by Multiple Herpes Simplex Virus Strains
More LessSUMMARYDistinct high frequency recurrence (HFRc) or low frequency recurrence (LFRc) phenotypes were observed following rabbit keratitis with three type 1 and five type 2 herpes simplex virus strains. LFRc strains were found to have latently infected the animal but were detected very rarely, if at all, in the eye following the acute phase. The recurrence phenotypes defined in singly infected animals remained unchanged following bilateral infection of the same animal with strains of opposite phenotype. Cocultivation of virus from bilaterally infected animals showed that both virus strains were capable of latently infecting the same animal. Restriction enzyme analysis of plaque-purified virus revealed that some ganglia were latently infected with both parental strains. Recombinants were also found. Some latently infected animals could be re-infected acutely. However, establishment of latency by the superinfecting strain was inhibited.
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Herpes Simplex Virus Defective Genomes: Structure of HSV-1 ANG Defective DNA of Class II and Encoded Polypeptides
More LessSUMMARYSequence organization and origin of HSV-1 strain Angelotti (ANG) class II defective DNA (HSV-1 ANG dDNA1) were examined in detail by establishing physical maps and by molecular cloning. dDNA1 consists of concatemers of tandem repeat units in which sequences from the U l region spanning map coordinates 0.37 to 0.415 of standard HSV ANG DNA are covalently linked to TR s /IR s sequences. The size of the repeat unit was determined to be about 8.9 kilobase pairs (kb), comprising sequences of 7.3 kb from U l and 1.6 kb from TR s /IR s regions. U l sequences were delineated by restriction enzyme sites KpnI N-P and EcoRI F-M, and were colinear with the corresponding sequences of the standard (wild-type) virus genome. Expression of dDNA1 was studied in African green monkey kidney cells and in Xenopus laevis oocytes. A major polypeptide of approx. mol. wt. 135000 (135K) was overproduced, suggesting that this protein was encoded by dDNA1. By several parameters, e.g. size, immune cross-reactivity, and affinity for native and denatured DNA, the 135K polypeptide was identified as the major HSV DNA-binding protein. It was further shown that the repeat unit contains part of the DNA polymerase gene as demonstrated by its ability to rescue some mutations in this gene.
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Accumulation of Herpes Simplex Virus Type 1 Glycoprotein D in Adhesion Areas of Infected Cells
More LessSUMMARYBy indirect immunofluorescence microscopy (IIF) we have localized glycoprotein D (gD) of herpes simplex virus-infected cells to the vinculin-containing junctional areas and to the focal adhesion sites on the ventral surface of the cells. Double-staining IIF showed that gD and vinculin co-distributed in the infected cells and treatment of the infected cells 4 h post-infection with rabbit antibodies to gD prevented attachment of the cells to the growth substratum. Our results therefore support the hypothesis that gD plays a central role in the social behaviour of infected cells.
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Preservation of Catecholamine Uptake and Release in Herpes Simplex Virus Type 1-infected PC12 Cells
More LessSUMMARYThe PC12 cell line, which is derived from a rat pheochromocytoma and possesses a number of ‘differentiated’ neuronal properties, was used to characterize the effect of herpes simplex virus type 1 (HSV-1) infection on the uptake and release of catecholamines. Both the uptake of [3H]norepinephrine and the content and K+-induced release of endogenous catecholamines were remarkably preserved during the course of productive infection. HSV-1 infection is thus selective in its effects on the host cell and certain specialized functional properties may be retained in the face of otherwise profound metabolic alterations.
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Reduction of 4-Nitroquinoline 1-Oxide to 4-Hydroxyaminoquinoline 1-Oxide in Lysates of Cytomegalovirus-infected Cells
SUMMARYThe rates of virus inactivation by 4-nitroquinoline 1-oxide (NQO) and 4-hydroxyaminoquinoline 1-oxide (HAQO) were compared and samples of cytomegalovirus (CMV)-infected cell lysates to which NQO had been added were examined for the presence of HAQO. These experiments demonstrated that (i) CMV inactivation by HAQO was more rapid than with NQO, (ii) virus inactivation by either NQO or HAQO failed to demonstrate a photodynamic component, and (iii) NQO-treated stocks contained HAQO, indicating reduction of NQO to HAQO. The results support the concept that metabolism of NQO to HAQO enhances the genotoxic effect of NQO.
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Cross-reactivity between Enteric Adenoviruses and Adenovirus Type 4: Analysis of Epitopes by Solid-phase Immune Electron Microscopy
More LessSUMMARYThe immunological relationships between the two newly discovered serotypes of enteric adenoviruses, Ad40 and Ad41, and antisera to human adenoviruses representing all subgroups were studied by solid-phase immune electron microscopy. A pronounced two-way cross-reaction was seen between Ad40 (subgroup F) and Ad41 (subgroup G). Furthermore, a distinct one-way cross-reaction was noted between adenovirus type 4 antiserum (subgroup E) and virions of Ad40.
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Distribution of Restriction Enzyme Sites in the Bovine Parvovirus Genome and Comparison to Other Autonomous Parvoviruses
More LessSUMMARYA physical map has been constructed for bovine parvovirus (BPV) replicative form (RF) DNA synthesized in vitro. Analysis of restriction enzyme digestion products for 12 enzymes by neutral gel electrophoresis allowed the mapping of 26 cleavage sites between the 3′ and 5′ termini of the in vitro RF DNA. Cleavage sites for these enzymes were not detected within the 3′-terminal hairpin. HindIII, SalI, SmaI, SstII and XorII did not cleave BPV DNA, while 12 other enzymes cleaved the genome at one to four sites. Eleven additional enzymes were shown to cleave the genome at 8 to 20 sites. Comparison of the restriction site locations for BPV with those of other autonomous parvoviruses suggests that there may be conservation within the genome of certain restriction sites.
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Characterization of the Genome of the Agent of Erythrocyte Aplasia Permits its Classification as a Human Parvovirus
More LessSUMMARYIt has recently been established that infection with a virus is the most common cause of transient arrest of erythrocyte production in the bone marrow, leading to aplastic crisis, in persons suffering chronic haemolytic anaemias. The physical characteristics of this human virus have suggested that it may be a member of the Parvoviridae. We report here that extraction of nucleic acid from this virus under annealing conditions yielded a single species of double-stranded DNA 5.5 kb in length. Treatment with heat or alkali converted this DNA into a rapidly migrating form sensitive to the single-strand-specific nuclease S1. Extraction of the virion DNA under conditions of low ionic strength where annealing would not be expected to occur yielded DNA which comigrated with the 5.5 kb single-stranded molecule. The results indicate that this virus packages equal numbers of complementary DNA strands into separate virions. It is suggested that this virus can be classified as a member of the genus parvovirus.
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Characterization of Monoclonal Antibodies to Potato Virus Y and Their Use for Virus Detection
P. Gugerli and P. FriesSUMMARYCultures of hybrid cells secreting monoclonal antibodies to potato virus Y (PVY) were produced by fusing a non-secreting FO myeloma cell line with spleen cells from BALB/c mice immunized with an isolate of the tobacco veinal necrosis strain group (PVY n 605). Monoclonal antibodies were obtained which reacted with common antigenic determinants of 24 isolates belonging to the homologous (PVY n ), common (PVY o ) and stipple streak (PVY c ) strain groups of PVY. Other antibodies were either strictly specific to PVY n isolates or of intermediate specificity. In enzyme-linked immunosorbent assay (ELISA) for the detection of PVY viruses, a preparation of monoclonal antibody to a common antigenic determinant gave more uniform reactions than polyclonal antibodies from rabbit serum. Enzyme conjugates of monoclonal antibody preparations could be used highly diluted without loss of activity in ELISA, thereby decreasing the background caused by non-specific reactions.
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Characterization of Viral-complementary RNA Associated with Polyribosomes from Tobacco Infected with Sonchus Yellow Net Virus
More LessSUMMARYPolyribosomal RNA from tobacco infected with sonchus yellow net virus was resolved by electrophoresis on agarose gels, transferred to nitrocellulose and hybridized to radioactive viral RNA. Polyadenylated RNA contained four major viral complementary species, molecular weights 2.2 × 106, 0.83 × 106, 0.62 × 106 and 0.46 × 106, plus a minor species, molecular weight approx. 3 × 106. No such species could be detected in the non-polyadenylated RNA fraction. The polyadenylated RNA from membrane-bound polyribosomes contained only the 0.83 × 106 and 0.46 × 106 molecular weight RNAs. The sizes of the RNAs were consistent with the expected sizes of messages for the virus proteins, calculated from the molecular weight of these proteins.
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